Novel cephalotaxane derivatives and process for their preparation

ABSTRACT

The present invention concerns a new general process for asymetric hemisynthesis of harringtonines and their analogs, that are alcaloïds used in chimiotherapy. This process comprises direct esterification of a natural cephalotaxine with an acylating compound constituted of a side chain precursor which backbone and fonctionalization are entirely preformed.

[0001] This application is a continuation-in-part of U.S. Ser. No. 09/270,006 filed Mar. 16, 1999, incorporated by reference in its entirety herein.

[0002] The present invention relates to a process for preparing cephalotaxane derivatives bearing a side chain.

[0003] The term “cephalotaxanes” refers to compounds or salts thereof which have a basic skeleton of formula

[0004] where p is equal to 1 or 2 (it being possible for the two units to be identical or different and linked via a single bond or an oxygen atom), which can contain various oxygenated substituents (aliphatic or aromatic ethers, free or esterified alcohols, substituted or free enols and/or phenols, bridged ethers, and more generally any substituent usually encountered in the natural state on compounds of this type).

[0005] Harringtonines are alkaloids which are of high interest in anticancer chemotherapy, in particular on certain haematosarcomas which are multi-resistant to the existing therapies. The selectivity of harringtonines, which is based on a novel mechanism of action relating to protein synthesis, is such that this series is favoured with a great future in anticancer therapy.

[0006] Several literature compilations give a seemingly exhaustive review of all of the knowledge relating to cephalotaxanes, these compilations being, chronologically: [C. R. Smith, Jr, R. G. Powell and K. L. Mikolajczack, Cancer Treat. Rep., Vol. 60, 1157 (1976); C. R. Smith, Jr, L. Kenneth, K. L. Mikolajczack and R. G. Powell in “Anticancer Agent Based on Natural Product Model”, 391 (1980); Liang Huang and Zhi Xue in “The Alkaloids”, Vol. XXIII (A. Brossi Ed.), 157 (1984); M. Suffness and G. A. Cordell in “The Alkaloids, Chemistry and Pharmacology” (A. Brossi Ed.), Vol. 25, 57-69, 295-298 (1987); P. J. O'Dwyer, S. A. King, D. F. Hoth, M. Suffness and B. Leyland-Jones, Journal of Clinical Oncology, 1563 (1986); T. Hudlicky, L. D. Kwart and J. W. Reed, in “Alkaloid: Chemical and Biological Perspectives” (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, 199 (1998)].

[0007] Antiparasitic activities, in particular on the haematozoon of malaria, have also been recognized [J. M. Whaun and N. D. Brown, Ann. Trop. Med. Par., Vol. 84, 229 (1990)].

[0008] Homo-harringtonine (HHT), the most active member of the series, is active at and above daily doses of 2.5 mg/m² of body area per 24 hours, i.e., as a guide, at doses twenty times lower than that for Taxol. HHT has already undergone fourteen phase I and II clinical trials and it is the only known product capable of a 70% reinduction of full haematological remissions in patients suffering from chronic myeloid leukaemias that have become resistant to alpha-interferon [S. O'Brien, H. Kantarjian, M. Keating, M. Beran, C. Koler, L. E. Robertson, J. Hester, M. Rios, M. Andreeff and M. Talpaz, Blood, 332 (1995); Leukemia Insights, Vol. 3, No. 1 (1998)].

[0009] Harringtonines were extracted over 35 years ago from an exclusively Asiatic cephalotaxacea known as Cephalotaxus harringtonia, following the programme of research into novel anticancer agents in the plant kingdom developed by the National Cancer Institute. In fact, the Cephalotaxus alkaloids consist essentially (at least 50%) of cephalotaxine, a biosynthetic precursor of the harringtonines, the latter individually representing only a few per cent of the total alkaloids.

[0010] Besides their low concentration in the natural state in plant starting material, harringtonines are mixed with many congeners which have very similar chemical structures. Thus, in a high resolution high performance liquid chromatography (HPLC) chromatogram of a semi-purified alkaloid extract, no less than several tens of cephalotaxine esters are counted.

[0011] If we consider that:

[0012] on the one hand, harringtonines are generally relatively non-crystallogenic, as is suggested by the flexibility of their side chains, which are generally branched and aliphatic,

[0013] on the other hand, these esters, in particular harringtonine and homo-harringtonine, are contaminated with congeners which are themselves biologically active and very difficult to separate out, even by high resolution analytical HPLC,

[0014] the current state of the art does not allow these compounds to be produced viably on the industrial scale as regards the purity required for pharmaceutical active principles.

[0015] Although biosynthetically similar to the alkaloids of the genus Erythrina, cephalotaxanes are alkaloids which have a unique structure in nature, encountered only in the genus Cephalotaxus, which is the only genus of the Cephalotaxacea family. On the other hand, the side chains of the various harringtonine congeners are all derived from the methyl hemiester of the primary carboxyl of (2R) citramalic acid 3a (see Scheme 1 attached) by substitution of the tertiary methyl using alkyl or aralkyl radicals which may themselves be unsubstituted or substituted with tertiary hydroxyls, it then being possible for the latter to form a cyclic ether with a tertiary alcohol (anhydro derivatives).

[0016] The attached Scheme 1 shows the main examples of harringtonine congeners, which all have significant cytostatic activity to different degrees. None of the artificial analogous cephalotaxine esters synthesized hitherto in the literature has at least the sub-structure 3b (see Scheme 1) and lack significant cytostatic activity.

[0017] It is worthwhile pointing out that, although botanically very similar to the Cephalotaxaceas, Taxaceas contain triterpene alkaloids (taxines), accompanied by non-alkaloid triterpenes, taxanes, which are also of unique structure in nature. Although they are completely different from taxanes in terms of chemical structures and anticancer mechanism of activity, the harringtonines have analogy with taxanes in more than one respect:

[0018] they have cytostatic properties,

[0019] they consist of a polycyclic skeleton, an inactive biosynthetic precursor of the complete structure, onto which is grafted a side chain containing a similar combination of hydrophilic and hydrophobic substituents,

[0020] the polycyclic part of the taxanes (baccatins in the broad sense) and of the harringtonines (cephalotaxines) is relatively abundant in renewable parts of the plant, whereas the active molecules (harringtonines and taxanes) are ten to one hundred times less abundant therein,

[0021] the plum yew (Cephalotaxus) is a rare tree, even rarer than the yew (Taxus), and is much less ubiquitous than the latter.

[0022] It results from the above facts that, following the manner of the semi-synthesis of taxanes by adding a synthetic chain to a 10-deacetylbaccatin III of extracted origin, the asymmetric semi-synthesis of harringtonines by esterification of a cephalotaxine of natural origin is of considerable medical and economic value. Furthermore, the current population of Cephalotaxus is relatively reduced even in their original habitat. Thus, during its importation into Europe for ornamental purposes last century, Cephalotaxus harringtonia was already no longer present in spontaneous form in eastern China and in northern Japan. The use of a precursor present in a renewable part of the tree (the leaf) in order to prepare homo-harringtonine semi-synthetically is thus of considerable environmental interest, all the more so since the total synthesis of optically active cephalotaxine has not been achieved hitherto, despite the extensive synthetic studies carried out in this respect (a certain number of laborious syntheses of racemic cephalotaxine containing 10 to 15 steps have, however, been carried out: see bibliographic review above).

[0023] Consider that several hundred tonnes per year of this rare and very slow-growing tree (even slower growing than Taxus sp.) need to be extracted to satisfy the current market needs for homo-harringtonine (several kilograms per year), whereas the semi-synthesis would consume only a few tonnes of renewable parts of the tree (leaves). Furthermore, homo-harringtonine (HHT) of natural origin currently available on the active principles market is contaminated with its congeners, which, on account of their structural similarity, are very difficult to separate, even by “preparative” high performance liquid chromatography.

[0024] First of all, it should be noted that since the use of cephalotaxine itself as a source for semi-synthesis has not yet been economically justified, no process for selectively extracting this substance has been described hitherto. Moreover, among the active compounds, only harringtonine and isoharringtonine have been the subject of American patent applications for their preparation by extraction [R. G. Powell et al., U.S. Pat. Nos. 3,793,454 and 3,870,727]. Harringtonine has been the subject of a Japanese patent [JP 58-032,880] and deoxyharringtonine has been the subject of an American patent [U.S. Pat. No. 3,959,312]. As regards the preparation of homo-harringtonine itself, it has been the subject of only a few semi-synthetic studies [T. Hudlicky, L. D. Kwart and J. W. Reed in “Alkaloid: Chemical and Biological Perspectives” (S.W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, 199 (1998)], but no patent application has been made regarding a semi-synthesis process or even an extraction process.

[0025] Another aspect which gives the present invention an even greater advantage is that cephalotaxine can serve as a springboard for the synthesis of cephalotaxoids and harringtoids which are useful for antitumour (cancerous and non-cancerous tumours), antiparasitic, antifungal, antiviral and antibacterial chemotherapies.

[0026] Harringtonines consist of a complex alkaloid polycyclic alcohol (cephalotaxine), esterified with a side chain, in isolation having no more biological activity than cephalotaxine, but essential for the biological activity of the whole. Saponification of the side chain under harsh conditions leads to the cephalotaxine free base and to harringtonic acids. The attachment of the side chains takes place at the end of the biosynthesis. It has been demonstrated that catabolism leading to this reaction could be triggered in vivo under the influence of environmental or physiological stress exerted on the plant [N. E. Delfel, Phytochemistry, 403 (1980)].

[0027] Cephalotaxine, the polycyclic part consisting of 5 fused rings, has a novel arrangement which is unique in nature, i.e. a benzodioxoazepine onto which is fused a spiropyrrolidinopentenediol system. Cephalotaxane contains four asymmetric centres: three “asymmetric carbons” and a heterocyclic tertiary aminic nitrogen. The only reactive function is a secondary alcohol located in position 3, the methyl enol ether located in position 2 being potentially sensitive to proton attack. The whole forms a pseudohelical structure encaging the hydroxyl in the tube formed by the tetrahydrazepine. The base cephalotaxine readily forms highly crystallogenic stable salts (for example hydrochlorides and perhydrochlorides).

[0028] This alkaloid is relatively insensitive to basic media. On the other hand, several authors describe a certain level of sensitivity to acids and to quaternization of the nitrogen with methyl iodide, leading to a racemization by simultaneous inversion of the 3 asymmetric centres and of the nitrogen [D. J. Abraham, R. D. Rosensten and E. L. McGandy, Tetrahedron Letters, 4085 (1969)]. However, a period of several days in solution at pH 1-4 at 20° C. leaves this structure intact (personal observation).

[0029] This compound and its congeners which are not O-acylated in position 3 are biologically inactive.

[0030] All the side chains for harringtonines which have significant biological activity contain in common the 2-alkyl-2-carbomethoxymethyl-2-hydroxyacetyl unit. The alkyl chain, of variable length, has at the end either branching constituting an isopropyl bearing (harringtonine HT and homo-harringtonine HHT) or not bearing (deoxy-homo-harringtonine DHT) a tertiary alcohol, or a phenyl radical (for example the neoharringtonine series most recently isolated). In the case of the anhydroharringtonines, the chain can be closed by dehydration between its two tertiary alcohols, for example forming a substituted tetrahydropyran ring. The tertiary carboxyl of this complex diester is borne by the single hydroxyl of the cephalotaxine. The only chiral centre on the side chain is located (to the ester junction. It contains, besides the first secondary chain, a hydroxyl which, on account of its tertiary nature, does not have the possibility of epimerizing.

[0031] Scheme 2 attached shows synthetically the known processes for preparing harringtonines.

[0032] Several semi-syntheses of natural cephalotaxine esters and several series of analogues, which have simplified chains but give these analogues reduced cytotoxic activity, have been described hitherto, in particular those of deoxyharringtonine and of isoharringtonine. Most of them relate to simpler and less functionalized esters than those constituting HT and HHT, the esters which are most useful in chemotherapy [for example, deoxyharringtonine, isoharringtonine, T. Hudlicky, L. D. Kwart and J. W. Reed in “Alkaloid: Chemical and Biological Perspectives” (S. W. Pelletier Ed.), Vol. 5, 639 (1987)].

[0033] All the literature from 1972 to the present date [Mikolajczack et al., Tetrahedron, 1995 (1972); T. Hudlicky, L. D. Kwart and J. W. Reed in “Alkaloid: Chemical and Biological Perspectives” (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, 5 T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, p. 236 (1998)] mention the impossibility hitherto of esterifying the highly sterically hindered secondary hydroxyl of cephalotaxane 2a with the tertiary carboxyl of the alkanoyl chain of harringtonic acid 3e totally preformed to give a harringtonine 4b, i.e. the conversion 2a+3e (4b as described in the example featured in the scheme below

[0034] Most of the syntheses described hitherto thus involve binding of the secondary side chain —CH₂CO₂Me, i.e.:

[0035] 1st) by the Reformatsky reaction between methyl bromoacetate and the carbonyl (real or potential) on the side chain prebound to cephalotaxine, in the presence of zinc, or

[0036] 2nd) by prior formation of an organolithium reagent.

[0037] All the syntheses described thus consist in esterifying cephalotaxine using the (-keto alkanoyl chloride 7 lacking the end hydroxyl and containing neither the secondary chain located (to the tertiary carboxyl, nor the tertiary hydroxyl (to the carboxyl, to give 8 which is then converted into a harringtonine 4a, according to the reaction described below.

[0038] In formula 8, CTX— represents the cephalotaxyl radical of formula:

[0039] It should be noted that this (-hydroxyalkylation, which at the same time creates the chiral centre on the side chain, has never been achieved asymmetrically.

[0040] A few synthetic routes involve an esterification of cephalotaxine with a substituted hemisuccinyl chloride, optionally followed by subsequent introduction of the tertiary hydroxyl(s).

[0041] No O-acylation of cephalotaxine, using totally preformed and functionalized chiral chain precursors (to the tertiary carboxyl, has thus been achieved hitherto [T. Hudlicky, L. D. Kwart and J. W. Reed in “Alkaloid: Chemical and Biological Perspectives” (S. W. Pelletier Ed.), Vol. 5, pages 661 to 675 (1987); M. A. Miah, T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, pages 224 to 236 (1998)].

[0042] Consequently, the methods for preparing harringtonines by semi-synthesis, which have been described to date in the existing art, have the following drawbacks:

[0043] absence of stereoselectivity,

[0044] poor convergence,

[0045] mediocre yields,

[0046] functionalization and construction of the chain on a rare and expensive substrate,

[0047] chiral homo-harringtonine not obtained to date.

[0048] Since cephalotaxine is present in nature in partially racemized form [personal observation; Huang et al., Scientia Sinica, Vol. XXIII, 835 (1980)], the processes of the prior art which use a natural cephalotaxine as starting material can only theoretically result in partially racemized harringtonines. The present invention thus has the advantage of obtaining enantiomerically pure harringtonines even from racemic cephalotaxine, since:

[0049] 1st) the asymmetric centre on the side chain is created prior to the esterification step, i.e. the side chain precursor can be obtained in enantiomerically pure form prior to being attached,

[0050] 2nd) the diastereoisomers obtained in the case of a racemic cephalotaxine can be separated by chromatography.

[0051] The present invention consists in:

[0052] esterifying the hindered free alcohol of a cephalotaxine or alternatively the corresponding metal alkoxide, using a chain in the form of a suitably substituted tertiary carboxylic oxacycloalkane acid which is totally preformed both in terms of the skeleton and in terms of the functionalization, in order to prepare anhydro-homo-harringtonic acids by semi-synthesis.

[0053] opening the cyclic side chains thus formed in order to obtain the corresponding diols, i.e. the harringtonines (defined above).

[0054] describing a new preparation for all of the diastereoisomers of the dihydroxylated side chains of the harringtonines in a dehydrated cyclic form (anhydroharringtonic acids) or in which the two hydroxyl groups are protected together by difunctional protecting groups forming a ring.

[0055] resolving all of the harringtonic and anhydroharringtonic acids, in order to couple them separately with the cephalotaxines.

[0056] One part of the present invention thus consists in synthesizing, in particular, anhydroharringtonine, harringtonine, anhydro-homo-harringtonine and homo-harringtonine.

[0057] The present invention also relates to esterifying cephalotaxines or metal alkoxides thereof with N-alkyl- and N-carbamoyl-2-alkylisoserine.

[0058] Following observations and comparative studies carried out in the taxane series, it was found that, despite a steric bulk which is still greater than that for the hydroxyl of the cephalotaxines, the hydroxyl located at position 13 on the taxane skeleton made it possible to receive acylation with a relatively bulky chain such as, for example, an N-benzoylphenylisoserine protected in position 2′ (although the attempts to acylate baccatin protected with a chain bearing an (hydroxyl group protected with a benzoyl group all failed).

[0059] An experimental study of acylation with a very bulky chain, such as pivalic acid, demonstrated the impossibility of acylating the hydroxyl located at position 13 of a baccatin protected by the dialkyl carbodiimide method, whereas the same reaction performed on cephalotaxine showed easy coupling of the pivaloyl chain.

[0060] It has also been demonstrated, again in the taxane series, that the improvement in the dynamic compactness of the alkanoyl or aralkanoyl chain by a bifunctional cyclic protection greatly facilitated the coupling. Thus, for example, French patent applications [J. P. Robin et al., FR 95/12739 and FR 95/15557] indicate that the suitably protected, linear N-benzoylphenylisoserines react in several hours at 80° C. with formation of epimerization products, the oxazoline or oxazolidine cyclic precursors reacted in less than one hour at 25° C.

[0061] The use of the same conditions as above with a chain which has undergone a prior dehydrating cyclization, allowed us to acylate the cephalotaxine or its alkoxides in a few hours at room temperature.

[0062] The ease of this acylation was all the more surprising since many authors have designed syntheses which have the drawbacks mentioned above, on the basis of the observation of an impossibility of acylating, explained by the steric hindrance at the two sites: the hydroxyl of the cephalotaxine and the tertiary carboxyl of the side chain precursor [Mikolajczack et al., Tetrahedron, 1995 (1972)].

[0063] According to a preferred embodiment or process according to the invention, the hydroxyl function of a cephalotaxane is esterified with a 2-carboxyl-2-alkyl-1-oxacycloalkane derivative.

[0064] The hydroxyl of a cephalotaxane whose skeleton corresponds to the general formula 1 or, more particularly, a secondary alcohol located in position 3 of a cephalotaxine or metal alkoxides thereof, corresponding respectively to the general formulae CTX—O—H and CTX—O—M, M being a metal and CTX— the cephalotaxyl radical defined above, are esterified with the tertiary carboxylic acid function of a substituted cycloether corresponding to the general formula of the type 3k, or alternatively, preferably, with one of its activated forms, isolated or formed in situ, corresponding to the general formula of the type 31 to give 4c, according to the reaction below:

[0065] R⁵, R⁶ and R⁸ are, independently of each other,

[0066] a hydrogen,

[0067] a linear or branched and/or cyclic, saturated, unsaturated or aromatic, hydrocarbon-based radical, in particular an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, an aryl, a heterocycloalkyl, the said hydrocarbon-based radical bearing or not bearing (a) heteroatom(s), it being possible for R⁵ and R⁸ to be linked together to form a ring

[0068] an oxygenated ether bearing one of the above radicals.

[0069] n is between 0 and 8.

[0070] A is a leaving group capable of leaving behind a carbocation, 31 can thus be, in particular: an acid halide, an anhydride, a mixed anhydride or a cyclic anhydride (when R⁵=—CH₂CO—).

[0071] The free acid of the type 3k or any activated form of the acids of the type 31 can be used to esterify the cephalotaxanes corresponding to the general formula 1 or, for example, the cephalotaxines of the type CTX—OH. Alternatively, the anhydride of 31 can be prepared separately and isolated, and then placed in contact with the alcohol. This is likewise the case for the cyclic anhydrides of the type 3p

[0072] in which n, R⁶ and R⁸ have the same meaning as above,

[0073] and which can be readily prepared from the corresponding diacids in order once again to give 3k by esterification of methanol or alternatively to become attached as above to the alcohol function of a cephalotaxine of the type 2 with, however, a poorer yield than that above, the primary acid function then being methylated conventionally using methanol in the presence of a protonic acid or a Lewis acid, or alternatively using the boron trifluoride etherate/methanol complex or diazomethane.

[0074] Although less effective and more laborious, the method using the acid chloride 3k gave the desired ester 4c.

[0075] All the reagents of the type 3k, 31 and 2, as well as the resulting esters of the type 4c, can be used alone in enantiomerically pure form, or in the form of a racemic mixture or in the form of diastereoisomeric mixtures. The intermediates can, in certain cases, not be isolated or formed in situ fleetingly.

[0076] The reaction can take place at between 0° C. and 140° C., with or without an organic solvent, it being possible for these solvents to be alone or as a mixture.

[0077] The esterification of the hydroxyl of cephalotaxane with a 2-carboxyl-2-alkyl-1-oxacycloalkane derivative can be carried out either by acyl transfer to the alcohol or by the carbodiimide method.

[0078] The esterification reaction by acyl transfer to the alcohol is advantageously carried out according to six specific modes:

[0079] (a) esterification of the free acid with the alcohol in acid catalysis,

[0080] (b) esterification by acyl transfer via anhydrides or halides,

[0081] (c) esterification by acyl transfer using activated esters,

[0082] (d) esterification with scandium triflate,

[0083] (e) esterification with boron trifluoride etherate,

[0084] (f) esterification by the thioester method.

[0085] The esterification (a) takes place by placing the acid of the type 3k and the alcohol of the type 2 in contact in solution in a co-solvent and in the presence of an acid catalyst. The displacement of the equilibrium can be promoted by adding a dehydrating agent or by azeotropic entrainment or by partition between two immiscible solvents, one of which is miscible with the ester formed and the other with water. These various methods can be combined.

[0086] The acid catalyst can be a protonic acid such as, for example, sulphuric acid, hydrochloric acid, boric acid, preferably para-toluenesulphonic acid, or a Lewis acid which may or may not be supported on a polymer, such as, for example, aluminium chloride, chlorotrimethylsilane or, preferably, boron trifluoride etherate. Advantageously, an ion-exchange resin or bisulphate on graphite can also be used, for example.

[0087] The dehydrating agent can be, for example, a dehydrating agent consisting of an inorganic salt which is low in water and inert with respect to the reaction, such as magnesium sulphate, sodium sulphate, calcium chloride or, preferably, a molecular sieve.

[0088] The esterification (a) uses the same reaction conditions as above. However, in order to result in the formation of the acylium ion characteristic of this method, the ordinary acid catalysts are replaced, for example, with anhydrous sulphuric acid or superacids such as, for example, hydrofluoric acid and its derivatives or antimony pentafluoride.

[0089] The esterification (b) consists in using the same physical operating conditions as above, but using 31, in which A represents a halogen (acid halide), another alkanoyl molecule which is identical (anhydride) or different (mixed anhydride), such as, for example, trifluoroacetyl, 2,4,6-trichlorobenzoyl, formyl, methoxyformyl, sulphonates, phosphates and chlorophosphate.

[0090] In a variant of the above method, and in the specific case in which R⁵=CH₂CO₂H, a cyclic anhydride of the type 3p can be used

[0091] in which n, R⁶and R⁸ have the same meaning as above,

[0092] which can be prepared very simply by treating the corresponding diacid with acetic anhydride, for example under the general operating conditions for the preparation of the mixed anhydrides described below.

[0093] The catalysis can be acidic, as indicated in the above method, or, preferably, alkaline, for example a tertiary base such as pyridine and/or dimethylaminopyridine (DMAP), pyrrolidinopyridine (PPY), triethylamine, or a stronger base such as a hydride, for example calcium hydride. The solvent can preferably be an aprotic solvent, for example hexane, toluene, dichloromethane, tetrahydrofuran and/or pyridine.

[0094] The esterification (c) is a method similar to the esterification (b). These ester preparation methods can also be advantageously used and involve, for example, 1-acyloxy-1,2,3-triazole or formamidinium or silyl ester or 2-acyloxypyridinium intermediates.

[0095] The carbonyldiimidazole method, in which an N-acylimidazole intermediate is involved, can also be used.

[0096] The carbodiimide method uses a dehydrating coupling agent such as a carbodiimide, for example dicyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbo-diimide (DIC) or 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.

[0097] The reaction can be catalysed with a tertiary base such as, for example, pyridine and/or dimethylaminopyridine (DMAP), pyrrolidinopyridine (PPY), triethylamine, 4-morpholinopyridine or any other substituted base. N-Hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (HO-Su) can also be used, for example. The molar ratio of 31 relative to 2 can be between 1/1 and 4/1.

[0098] The reaction can preferably be carried out under inert gas at a pressure close to atmospheric pressure, preferably at a temperature of between 0° C. and about 110° C.

[0099] The solvents preferably used are organic solvents such as, for example, toluene and/or dichloromethane and/or chloroform and/or tetrahydrofuran and/or pyridine and/or dimethylformamide.

[0100] The application of the methods described above, for the substituted carboxylic oxacycloalkanes, to the coupling of their synthetic precursors, the (linear) 1-hydroxy-1-methoxycarbonylmethylalkenecarboxylic acids, made it possible, against all expectation, to synthesize the esters of the type 4c in a single step from ethylenic tertiary (-hydroxy acids of the type 3f instead of the sequence 3f (3k(3(4c.

[0101] where m is between 0 and 3,

[0102] in formula 4c, n, R⁵, R⁶, R⁸ and CTX— have the same meaning as above.

[0103] Indeed, the ethylenic tertiary (-hydroxy acid 3f treated under conditions similar to those above directly gave the cyclic ester of cephalotaxines of the type 4c without isolation of an intermediate. In the case of the method involving a mixed anhydride 3l, the in situ formation of the corresponding (-lactone has been assumed on account of the presence of an infrared band at 1840 cm⁻¹.

[0104] These elements were confirmed by the formation and isolation of 3l, using 3f only under the activation conditions described above, i.e. to form the mixed anhydride, for example in the presence of 2,4,6-trichlorobenzoyl chloride, or alternatively, for example, in the so-called DCC method mentioned above.

[0105] The substituted carboxylic cycloethers of the type 3k, the substituted ethylenic tertiary (-hydroxy acids of the type 3f, their activated intermediates, and cyclic anhydrides of the type 3p, can be coupled with the cephalotaxines, either in the racemic series, or, more advantageously, in the optically active series.

[0106] In the case of coupling between one of the above types of acid, in the form of racemic mixtures, with a single enantiomer of a cephalotaxine, a relative stereoselectivity has been found due to the chirality and the major steric hindrance of the reaction site, in the sense that the ratio between each of the two diastereoisomers is generally other than 1.

[0107] The separation of the two diastereoisomers formed of the type 4c (R⁵=CH₂CO₂Me), known as “anhydroharringtonine”

[0108] in which n, R⁶, R⁸ and CTX— have the same meaning as above,

[0109] can be carried out by preparative chromatography either in a so-called normal phase, for example on native silica gel as stationary phase and a mixture of organic solvents as mobile phase, or, preferably, in a reverse phase, for example an inert silica grafted with apolar groups such as, for example, organosilyl, cyanoalkyl, phenylalkyl, preferably ocatadecylalkylsilane, chains and a mixture of aqueous solvents as mobile phase.

[0110] In the case of an enantiospecific coupling, no trace of epimerization is observed on any of the original parts, and the only diastereoisomer obtained can be crystallized. When this diastereoisomer is not crystalline, it is chromatographed by flash chromatography in order to remove the reagent residues, and is then precipitated by addition to a non-solvent, in order to be isolated in the form of amorphous powder.

[0111] The opening of the cephalotaxine carboxylate cycloethers of the type 4c to give the halo alcohol 4d is shown in the following scheme.

[0112] In these formulae n, R⁵, R⁶, R⁸ and CTX— have the same meaning as above, X being a heteroatom such as a halogen.

[0113] The cyclic ethers of the type 4c can, in certain examples, have the particular feature of simultaneously containing a methyl enol ether, for example in position 2 of the cephalotaxines. Despite the usual inertia of true cyclic ethers (i.e. non-hemiacetal ethers), the placing in contact of a solution of 4c in an organic solvent, preferably a chlorinated solvent such as, for example, dichloromethane, dichloroethane or chloroform, under controlled conditions, i.e. in the presence, for example, of a dilute hydrohalic acid, preferably hydrobromic acid in acetic acid, at low temperature, or else in the presence of a halo-trialkylsilane or alternatively of a boron trihalide, for example boron tribromide at low temperature in an organic solvent, preferably a chlorinated solvent, such as, for example, dichloromethane, dichloroethane or chloroform, allowed it to be selectively opened to give a halo alcohol of the type 4d such that X=halogen, with a quantitative yield without any appreciable formation of the O-demethylation product, even in the case of cephalotaxines bearing enol ether(s) or other functions sensitive to ether-cleaving agents. In any case, in the event of an accidental demethylation, the enol can easily be selectively remethylated as described in the literature (for example by dissolution in methanol in the presence of para-toluenesulphonic acid).

[0114] The halo alcohols of the type 4d such that X=halogen are of great interest as substrates necessarily leading to heteroatomic analogues, on account of the very good reactivity towards the halogen substitution they bear.

[0115] Another variant consists in placing the product of type 4c in contact with an aqueous acid optionally in a miscible or immiscible co-solvent. The acid can be, for example, protonic, and in this case it is an organic or inorganic acid, preferably hydrochloric, tetrafluoroboric or formic acid. In this case, the diols of the type 4b can be isolated directly without passing through the halo alcohol stage described above.

[0116] The halo alcohols of the type 4d such that X halogen are hydrolysed according to the following scheme:

[0117] in which n, R⁶, R⁸, R⁵, X and CTX— have the same meaning as above.

[0118] The halo alcohols of the type 4d are particularly suitable for controlled hydrolysis by placing in contact with an aqueous inorganic base such as, for example, dilute sodium hydroxide, sodium carbonate or, preferably, barium carbonate, at a temperature of between 0° C. and 30° C. with stirring, to give the diols of the type 4b.

[0119] One variant consists in carrying out the in situ hydrolysis of the intermediate halo alcohols of the type 4d at the end of the reaction, preferably carried out by treating the cyclic ether of the type 4c in dichloromethane in the presence of hydrobromic acid in acetic acid, and by directly adding the water or the hydrolysis solution to the reaction medium while cold and with vigorous stirring.

[0120] The process according to the invention is particularly suitable for the preparation of azaharringtonines, nitrogenous analogues of the harringtonines.

[0121] In order to demonstrate, for example, the flexibility of use of halo alcohols of the type 4d such that X=halogen as substrates, these materials were, for example, subjected to azidolysis by treatment using, for example, an alkaline azide in a solvent such as, for example, ethanol, methanol or dimethylformamide, which, by hydrogenolysis in solution in an organic solvent such as, for example, an alkanol or a lower ester, lead to the corresponding amino alcohol corresponding to the formula 4f (aminodeoxyharrington-ine). The amine can then be subjected to amidation under the Schotten-Baumann conditions, i.e. in aqueous media in the presence of an inorganic base as catalyst, to give an amido alcohol of the type 4

[0122] such that Z=NHCOR or NHCOAr, R and Ar being as defined above, more specifically alkyl or aryl groups, respectively, which may or may not be substituted. The amine 4f can also be sec-alkylated to give an alkylaminodeoxyharringtonine (Z=NHR or Z=NHAr or Z=NR₂ or Z=NHAr₂, it being possible for the two radicals R and Ar to be identical or independent), or acylated to give amides (Z=NHCOR or Z=NHCOAr) or carbamates (Z=NHCOOR) derived from the corresponding aminodeoxyharringtonine 4f,

[0123] n, R⁵, R⁶, R⁸, R⁹, X and CTX— being defined as above.

[0124] Alternatively, the cyclic ethers of the type 4c can be suitable for the Ritter reaction in the presence of a nitrile (which can serve as solvent) at a low temperature of between −100° C. and +30° C. in the presence of an acid such as sulphuric acid, perchloric acid or, preferably, tetrafluoroboric acid, to give an acylaminodeoxyharringtonine (Z=NHCOR or Z=NHCOAr) derived from the corresponding aminodeoxyharringtonine 4f.

[0125] The extraction of the cephalotaxines of the type 2 is carried out according to the procedure indicated below.

[0126] The cephalotaxines of the type 2 can be prepared according to the methods described in the literature, either by synthesis or by extraction. In the latter case, since no method uses a direct placing in contact of the plant starting material with an aqueous acid, it has been found to be advantageous to describe this in the present invention. The fresh or dry plant starting material is placed in contact for 24 h with an acidified aqueous-organic mixture using a dilute inorganic acid or a weak organic acid, so as to bring the pH to between 1 and 4, preferably 3. The inorganic acid can be, for example, sulphuric acid or hydrochloric acid and the organic acid can be citric acid, lactic acid or tartaric acid, for example; the organic solvent can be, for example, a lower alkanol, a ketone, tetrahydrofuran or any other water-miscible solvent used in extraction by those skilled in the art. The water content is between 20 and 80%, preferably 50%. The solution obtained can be directly chromatographed or basified in order to be counter-extracted, since, in contrast with the methods described in the literature, it contains no chlorophyll and/or plant fat. The counter-extraction using a water-immiscible organic solvent such as a lower ester or, preferably, a lower halogenated hydrocarbon, more particularly dichloromethane, gives a mixture of total alkaloids isolated in the form of a white powder. Several methods for purifying Cephalotaxus alkaloids exist, but none, in particular in reverse phase, is specifically geared towards the purification of cephalotaxines and more particularly of the cephalotaxine of formula 2a.

[0127] The present process for purifying cephalotaxine, which forms an integral part of the novel process for the semi-synthesis of harringtonines, thus involves reverse-phase chromatography, which has never been used for this purpose. This reverse-phase chromatography uses as stationary phase, for example, an inert silica grafted with apolar groups such as, for example, organosilyl, cyanoalkyl, phenylalkyl or, preferably, octadecylalkylsilane chains such as those encountered commercially, and a mixture of aqueous solvents as mobile phase, preferably water itself (without organic solvent); the pH is adjusted, i.e. to between 2 and 4, with an inorganic acid such as hydrochloric or phosphoric or sulphuric acid. It is also advantageous to add an additive such as, for example, aqueous ammonia or triethylamine. According to this process, which is economically very advantageous since it avoids the use of organic solvent and allows the reuse of the stationary phase for virtually hundreds of operations, the cephalotaxine is obtained in a quantitative recovery yield and with a purity of greater than 95%.

[0128] The above method makes it possible to obtain not only laevorotatory cephalotaxines naturally present in the plant material, but also racemic cephalotaxines also present in the natural state.

[0129] The metal alkoxides, corresponding to the general formula 1b (n=1 to 12) in which M is a metal, more particularly an alkali metal such as sodium, potassium or lithium, or a transition metal, for example zirconium, titanium or zinc, can be obtained by metallation of one or more of the hydroxyls in the mono- or polyhydroxycephalotaxanes corresponding to the general formula 1a (x=1 to 12) and in which M is more particularly an alkali metal or alkaline earth metal or any other metal which can conventionally give rise to the formation of an alkoxide.

[0130] This formation of alkoxide is of great value for acylating more readily in this form the hindered hydroxyls of the cephalotaxanes and most particularly for coupling this cephalotaxane with acylating precursors of the side chains, automatically leading to the harringtonines which are the subject of the present invention.

[0131] Several methods can be used to metallate the hydroxyl(s) of a cephalotaxane. For example, a metal hydride, an alkylmetal, an amide or, more generally, any agent capable of exchanging or of giving up a metal atom can be used.

[0132] The simple placing in contact of a hydroxylated cephalotaxane in organic solution, preferably under an inert gas, with a metal hydride such as, for example, potassium hydride, lithium hydride or, more particularly, sodium hydride, leads to a cephalotaxane metal alkoxide, which can, for example, serve as an in situ substrate in order to attach, for example, a suitably substituted alkyl, acyl or alkylsilyl group. The organic solvent can be a suitable aprotic solvent such as an ether, more particularly tetrahydrofuran, a liquid aromatic hydrocarbon, preferably toluene or, more generally, any organic solvent which is liquid under the temperature and pressure conditions used and which has no appreciable reactivity towards the reagent. The temperature of the reaction medium can be between −90° C. and +30° C.

[0133] The simple placing in contact of a hydroxylated cephalotaxane in organic solution, preferably under inert gas, with a metallated hydrocarbon such as, for example, a lithiated hydrocarbon, preferably butyllithium, leads to the same metal alkoxides as those above. The same solvents as above can be used, except that, since the reactivity of the metal hydrocarbons is generally greater than that of the metal hydrides, the temperature is between −100° C. and −20° C., preferably between −60° C. and −80° C.

[0134] The simple placing in contact of a hydroxylated cephalotaxane in organic solution, preferably under inert gas, with an amide, preferably an alkali metal amide, for example an alkali metal dialkylamide such as lithium dicyclohexylamide or lithium diisopropylamide or alkali metal (lithium, potassium or sodium) bis(trialkylsilyl)amide leads to the same metal alkoxides as above. The same solvents as above can be used.

[0135] As an example and without detracting from the generality of the present invention, the cephalotaxine 2a in solution stirred in tetrahydrofuran at −70° C., treated with one equivalent of butyllithium or lithium bis(trimethylsilyl)amide leads, in a few hours, to the lithium alkoxide 2h, which, when trapped in situ with acetic anhydride, gives the 3-O-acetylcephalotaxine 2b.

[0136] The preparation of the substituted racemic carboxylic cycloethers of the type 3k is detailed below.

[0137] According to a first variant, the preparation of these carboxylic cycloethers can be carried out by cyclization of the substituted ethylenic tertiary (-hydroxy acids of the type 3f according to the following scheme:

[0138] m, R, R⁵, R⁶, R⁸ and A being defined as above.

[0139] The substituted carboxylic cycloethers of the type 3k such as, for example, A or B of formulae:

[0140] can be prepared from the substituted ethylenic tertiary (-hydroxy acids of the type 3f, by simple dissolution in an organic solvent in the presence of an acid.

[0141] According to a second variant, the preparation of these carboxylic cycloethers can be carried out by cyclization concomitant with the formation of the acylating species. As mentioned above, acids whose tertiary alcohol is free, such as 3f, cyclize spontaneously by the action of a dehydrating agent required for a certain technique for acylating a cephalotaxine alcohol mentioned above and then esterifying the latter to give 4c.

[0142] In the absence of an alcoholic substrate to receive it, the ethylenic tertiary alcohol of the 3f type leads, under anhydrous operating conditions, to the isolation of the acylating intermediate 3, mentioned above, or, by hydrolysis, to the isolation of the acids of the type 3k.

[0143] In this case, the procedure used is strictly the one described for coupling involving the formation of an acylating species in situ but in the absence of a substrate of the cephalotaxine type.

[0144] According to a third variant, these substituted carboxylic cycloethers of the type 3k can be prepared by deprotecting the tertiary carboxyl of the suitably substituted precursor 3h,

[0145] R⁵, R⁸, R⁶ and n being defined as above and GP representing a protecting group for the acids, with, as a specific case, GP=R.

[0146] According to a fourth variant, in the specific case in which R⁵=CH₂CO₂R, the suitably substituted carboxylic cycloethers of the type 3k such that R⁵CH₂CO₂R below,

[0147] R⁸, R⁶ and n being defined as above,

[0148] can be prepared by total saponification of the corresponding diesters 3j such that R⁵=CH₂CO₂R, followed by mild selective methylation of the intermediate diacid 3r such that R⁵=CH₂CO₂H.

[0149] Incidentally, this process, carried out at room temperature and with rigorous monitoring of the reaction kinetics, leads to selective saponification of the above primary ester; it thus gives access to derivatives of the type 3t such that R=GP below,

[0150] R⁸, R⁶, GP and n being defined as above,

[0151] which can be coupled with the cephalotaxines using the methods described above in order to ensure the absence of transesterification during the coupling of the tertiary acids which form the subject of the present invention.

[0152] According to a fifth variant, the preparation of these carboxylic cycloethers can be carried out by regioselective methanolysis of the corresponding cyclic anhydride.

[0153] As mentioned above, the diacid 3r leads, by self-dehydration, to the cyclic anhydride 3p, which is a good acylating agent for alcohols, such that by methanolysis 3k is also preferentially obtained such that R⁵=CH₂CO₂Me, which constitutes an additional preparation method.

[0154] The preparation of the substituted ethylenic tertiary (-hydroxy acids of the type 3f is detailed below.

[0155] The substituted ethylenic tertiary (-hydroxy diacids which are the precursors of the monoacids of the type 3f can, like their cyclic analogues of the type 3k above, be obtained:

[0156] either by selective deprotection of their precursors of the type 3i

[0157] or, for example, in the case of the diacids of the type 3s such that R⁵=CH₂CO₂H,

[0158] by selective methylation of the primary carboxyl in particular, by placing the reactants in prolonged contact in methanolic solution at room temperature or using the boron trifluoride/methanol complex.

[0159] The diacids of the type 3s such that R⁵=CH₂CO₂H can be obtained by saponification of the corresponding diesters of the type 3g such that R⁵=CH₂CO₂Me by placing the latter in contact with an excess of base in an aqueous or aqueous-alcoholic medium,

[0160] m, R, R⁸ and R⁶ being defined as above.

[0161] The alcohol can be a lower alcohol such as methanol, ethanol or, preferably, isopropanol, and the base can be, for example, an alkali metal or alkaline earth metal base or a rare earth metal hydroxide or aqueous ammonia. When the reaction takes place at a temperature of between 0° C. and 30° C. for 15 minutes to 1 hour, the regioselective saponification of the primary ester can be obtained without any resulting difficulty. By increasing the temperature to the boiling point of the solvent mixture and/or by lengthening the reaction time, the diacid is obtained in good yield and without formation of by-products.

[0162] The diacids of the type 3s such that R⁵=CH₂CO₂H can, on account of their crystallogenic properties, then generally be obtained in enantiomerically pure form by successive crystallizations of enantiomerically enriched mixtures until a constant optical rotation is obtained.

[0163] The diacids obtained above can then be converted into substituted ethylenic tertiary (-hydroxy acids of the type 3f such that R⁵=CH₂CO₂Me by selective mono-esterification of their primary carboxyl, using methanol in the presence of a protonic acid or a Lewis acid or alternatively using the boron trifluoride etherate/methanol complex or diazomethane.

[0164] Incidentally, and as for the cyclic analogues of the type 3t such that R⁵=CH₂CO₂H, above, this process carried out at room temperature and with rigorous monitoring of the reaction kinetics leads to selective saponification of the above primary ester; it thus gives access to derivatives of the type 3u such that R⁵=CH₂CO₂H, which can be coupled to the cephalotaxines using the methods described above in order to ensure the absence of transesterification during the coupling of the tertiary acids which form the subject of the present invention.

[0165] The substituted ethylenic tertiary (-hydroxy esters of the type 3g can be prepared according to the scheme outlined below:

[0166] R⁶, R⁸, m and R⁵ having the same meaning as above.

[0167] The ethylenic esters of the type 3g can be prepared according to the numerous methods described in the literature for similar cases such as, for example, (-hydroxyalkylation of the corresponding 1-alkyl- or 1-alkenyl-1-keto ester of the type 9.

[0168] As an example and without removing anything from the generality of the present invention, the (-hydroxyalkylation of the 1-alkenyl-1-keto ester of the type 9 with the lithium methoxycarbonylmethyl enolate (R⁵M=MeOCOCH₂Li)or of the corresponding organozinc reagent (Reformatsky reaction, in which R⁵MX=MeOCOCH₂ZnBr) leads to the diester 3g such that R⁵=CH₂CO₂Me.

[0169] The same reactions applied to a chiral ester (R=R*) lead to a mixture of separable diastereoisomers which, after deprotection of the tertiary acid function, each lead to the diastereoisomer of the pair.

[0170] Moreover, the (-hydroxyalkylation reaction of the 1-alkyl- or 1-alkenyl-1-keto ester of the type 9, conducted in the presence of a chiral inducing agent such as sparteine or quinine, can give a significant enantiomeric enrichment, which can be further enhanced by fractional crystallization.

[0171] The keto esters of the type 9 are themselves conventionally obtained by C-semi-acylation of the carbanion of the corresponding alkyl or alkenyl halides of the type 10 with a dialkyl oxalate.

[0172] One of the advantages over the prior art of the synthetic process which forms the subject of the present invention lies in the possibility of coupling an entirely preformed chain with the cephalotaxines. Thus, the preparation of the above anhydroharringtonic acids in enantiomerically pure form 3k is of considerable interest, since the post-coupling creation of the chiral centre in position 2′ of the harringtonines during the attachment of the secondary chain as described in the prior art leads to an epimeric mixture, on the one hand, which is very difficult to separate, and, on the other hand, to a loss of about 50% of the very precious cephalotaxines (not recyclable in a process for manufacturing a medicinal substance using Good Manufacturing Practice).

[0173] Several methods have been used to achieve this aim. They all apply both to the cyclic monoacids of the type 3k or to their diacid precursors of the type 3r, and to their ethylenic linear precursors of the type 3f, it being possible for chiral chromatography methods also to be applied to the precursors which have no function capable of engaging a reversible chemical bond with a chiral species (in this instance free acid functions).

[0174] According to a first step of the process for the enantiospecific preparation of these acids, an epimeric mixture is formed by combination with a chiral alcohol or amine.

[0175] The reactions for esterifying a hindered secondary alcohol function of a cephalotaxine with oxacycloalkanecarboxylic acids of the type 3k above (including those formed in situ from ethylenic tertiary (-hydroxy acids of the type 3f) can also be applied to the esterification of another chiral alcohol in order to convert a racemic mixture, or one with partial enantiomeric enrichment of acids of the type 3k, into a diastereoisomeric mixture on which all of the non-chiral separation methods become applicable. The above methods are also applicable without modification to the amidation of chiral primary or secondary amines.

[0176] Thus, when the oxacycloalkanecarboxylic acids of the type 3k or their ethylenic linear precursors are reacted with a chiral alcohol, denoted by R*OH, or an amide R′*R*NH (it being possible for R′* to be replaced with a hydrogen), two chemical species are obtained in which the physicochemical properties are distinct (for example NMR, melting point, solubility, chromatographic properties, enzymatic or microbiological attack, etc.). The alcohol or the amide must preferably be hindered and bear their chiral centre at their site of binding with the tertiary carboxyl of the acid of the type 3k. The alcohol can be, for example, menthol, borneol, valinol or, preferably, quinine. The amine can be, for example, ephedrine; more generally, any commercial chiral alcohol or amide can be used.

[0177] n, R⁵, R⁶, R⁸ and A having the same meaning as above, R* having the same meaning as R, but being chiral.

[0178] As an example, and without detracting at all from the generality of the present invention, (−)-quinine, which, like the cephalotaxines, is an alkaloid with a sterically hindered secondary alcohol function, reacts with the racemic mixture of the oxacycloalkanecarboxylic acids of the type 3k to give the mixture of the two corresponding epimers 3v such that R⁵=CH₂CO₂Me and 3w such that R⁵=CH₂CO₂Me:

[0179] The binary mixtures of epimers obtained by the combination with a chiral compound can be separated, for example, by fractional crystallization, by distillation, by counter-current liquid-liquid partition and, given the high added value of these intermediates, by any common preparative chromatography technique, for example normal phase chromatography, exclusion chromatography, preferably in reverse or normal grafted phase. Since these methods are synergistic, they can advantageously be combined in order to improve the diastereoisomeric purity.

[0180] As an example and without detracting at all from the generality of the present invention, the mixture of the two epimers 3v (such that n=3; R⁶=R⁸=Me; R⁵=CH₂CO₂Me) and 3w (such that n=3; R⁶=R⁸=Me; R⁵=CH₂CO₂Me), cited in the above example, can be separated without difficulty and in quantitative yield using a grafted phase of octadecylsilane type and a methanol/water mobile phase.

[0181] The regeneration of the oxacycloalkane-carboxylic acids of the type 3k in enantiomerically pure form can be carried out by total hydrolysis followed by selective remethylation of the primary carboxyl of the suitably selected diastereoisomer (see above sequence 3j (3k) or, when it is an ester bond with an oxygen in the benzyl position (see for example quinine above), by simple hydrogenolysis. In the latter type of case, the drawback of the hydrogenolysis is largely offset by the economy of a step on an expensive product. As an example and without detracting at all from the generality of the present invention, (−)-quinine (2′R)-anhydroharringtonate 3v (such that n=3; R⁶=R⁸=Me; R⁵=CH₂CO₂Me) gave a (2R)-anhydroharringtonic acid of the type 3k and dihydrodeoxyquinine which can thus not be recycled, but this is a minor drawback in view of the low cost of this akaloid. Alternatively, the double saponification of 3v followed by selective remethylation gave a product which was entirely identical to the (2R)-anhydroharringtonic acid of the type 3k above.

[0182] The enantiomer of non-natural configuration (2S) can, after having undergone the same conversions as its (2R) enantiomer, be exploited, for example, for the purposes of structure-activity relationship studies.

[0183] According to a first step of the process for the enantiospecific preparation of these acids, the racemic mixtures are resolved by formation of salts with a chiral basic species.

[0184] The racemic mixtures of oxacycloalkane carboxylic acids of the type 3k (including those formed in situ from ethylenic tertiary (-hydroxy acids of the type 3f), can form a salt with a chiral amine by simple placing together in solution in an organic solvent. Although most of the methods described above for separating the esters and amides formed with 3k are applicable (for example chromatography), since the salts formed are generally highly crystallogenic, it is fractional crystallization which is preferably carried out to resolve the acids of the type 3k. The solvents used, alone or as a mixture, can preferably be polar organic solvents which may or may not be combined with water, such as, for example, ketones, alcohols and lower esters. The reaction to form the salt preferably takes place at a temperature of between 0 and 100° C. The recrystallization can be carried out by redissolving the salt in a mixture whose solvent power can be adjusted with precision by means of the use of the above combinations of solvents and by varying the temperature according to the standard techniques practised by those skilled in the art. When the diastereoisomeric enrichment is deemed to be sufficient, the salt is decomposed in the presence, for example, of a dilute aqueous acid such as hydrochloric acid or sulphuric acid. The extraction of the enantiomer of the regenerated acid can be carried out using a water-immiscible organic solvent such as, for example, a lower ester.

[0185] As an example and without detracting at all from the generality of the present invention, the racemic mixture of (2R or 2S)-anhydroharringtonic acids of the type 3k can be resolved, for example, by placing them in contact with (−)-ephedrine, followed by fractional recrystallization in an ethyl acetate/methanol mixture.

[0186] The 2R-anhydroharringtonic acid of the type 3k is then regenerated by placing the purified salt in contact with 2N hydrochloric acid and continuously extracting the acidic aqueous phase with ethyl acetate.

[0187] The oxacycloalkanecarboxylic acids of the type 3k described above can be subjected to preparative chiral chromatography.

[0188] The final products are purified by HPLC to give final products for pharmaceutical use.

[0189] Despite the performance levels of the modern methods of synthesis, of semi-synthesis and of isolation of natural substances, it is now established in the regulations issued by the health authorities in industrialized countries that impurity levels of greater than one per thousand (0.1% m/m) in a medicinal substance can be detrimental to the patient.

[0190] An identification followed by toxicology studies on any toxicologically unknown substance exceeding this threshold is, moreover, systematically demanded, in order to obtain pharmaceutical files for authorization to market the medicinal products.

[0191] The diastereoisomeric purity (with, as a specific case, the enantiomeric purity) can, moreover, lead to therapeutic aberrations; for example, it is well known that quinine (see above formula) is an antimalaria agent, whereas one of its diastereoisomers is a cardiac antifibrillant.

[0192] In the therapeutic field of the substances forming the subject of the present invention, it is common to encounter multiplications of from 10 to 100 of the active principle or of a side effect by minor changes (involuntary here) in the molecular structures.

[0193] Among the methods for achieving this level of purity, industrial high-resolution chromatography occupies a position of choice, its high cost being an argument which carries little weight compared with the very high added value of the sophisticated active principles, the robustness which it gives to the processes and the safety it offers to users.

[0194] As an example, and without detracting at all from the generality of the present invention, homo-harringtonine 4b, such that n=3; R⁶=R⁸=Me; R⁵=CH₂CO₂Me, CTX—=cephalotaxyl,

[0195] R⁶, R⁸, R⁵, n and CTX— being defined as above,

[0196] can be freed of its epimer at the same time as its other related impurities by preparative reverse-phase chromatography using a grafted reverse phase of octadecylsilane type as stationary phase and a suitably adjusted methanol/water mixture as mobile phase.

[0197] This process gives a product whose sum of related impurities is less than 0.5% and for which none of these impurities taken individually exceeds 0.1%.

[0198] The present invention concerns a process for the preparation of sidechain-bearing cephalotaxane of the following formula and/or a salt thereof

ΩCO—O—CTX

[0199] where

[0200] Ω (“omega”) is a representative radical of the chain terminal moiety and —CO— is the carbonyl of the ester group bonded to cephalotaxane;

[0201] the Ω—CO— radical is corresponding:

[0202] either to the following substituted heterocycloalkane formula:

[0203] where n is included between 0 and 8;

[0204] Z is oxygen, nitrogen or sulfur heteroatom;

[0205] R⁵, R⁶ and R⁸ are independently

[0206] hydrogen;

[0207] hydrocarbon radical, saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl, of said radical including or not heteroatom(s); R⁶ and R⁸ may be included in a cycle;

[0208] oxygen ether bearing one of the former radicals;

[0209] or to the following linear alkene formula:

[0210] where m is included between 1 and 8, R⁵, R⁶ and R⁸ are as defined above;

[0211] or to the following formula:

[0212] where n, R⁵, R⁶ and R⁸ are as defined above;

[0213] Z and Q² are independently oxygen, nitrogen or sulfur heteroatom;

[0214] Q1 is carbon, silicium or phosphorus atom;

[0215] R⁹ and R¹⁰ are independently hydrogen, alkoxy, hydrocarbon radical, including or not heteroatom(s), saturated, unsaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl;

[0216] R⁹ and/or R¹⁰ having the ability to be null or taken together to make an heteroatom and/or make a multiple bond with Q1, R⁹ and R¹¹ having the ability to be null to make a multiple bond between the two atoms of carbon bearing them; and

[0217] R¹¹ is hydrogen, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl or alkylcarbonyl;

[0218] where

[0219] —O—CTX is cephalotaxine moiety of the following formula a salt thereof:

[0220] where p is equal to 1 or 2;

[0221] the two types of radicals —W and —CTX above-mentioned being bonded with an ester bond —CO—O—

[0222] the said process bringing together:

[0223] either carboxylic acid with general formula W—CO—OH or a salt thereof;

[0224] or an activated form of an acid with general formula W—CO—A or a salt thereof,

[0225] with W—CO of the following formula:

[0226] where n, Z, R⁵, R⁶ and R⁸ are as defined above;

[0227] where W—CO of the following formula:

[0228] m is included between 1 and 8, Z, R⁵, R⁶ and R⁸ are as defined above; where W—CO of the following formula:

[0229] and where n, Z, Q¹, Q², R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined above

[0230] A represents:

[0231] either cyclic anhydride of the following formula:

[0232] where n, R⁶and R⁸ are as defined above;

[0233] the reaction has been completed by methylation of the primary carboxyl thus formed, with:

[0234] either a cephalotaxane or a salt thereof, bearing at least a free hydroxyl group, of the formula H—O—CTX, where CTX are as defined above;

[0235] or a metallic alcoxide of the formula M—O—CTX, where CTX are as defined above and M is a metal;

[0236] or an activated form of its hydroxyl group of the formula Y—O—CTX, where —O—CTX is as defined above and Y is, either a leaving group to allow a negative charge on oxygen atom by cleavage between Y— and —O—CTX, or to allow a carbocation by cleavage between Y—O— and —CTX;

[0237] with the possible presence of one or several reaction additives to form said sidechain-bearing cephalotaxane and/or a salt thereof.

[0238] Most preferably, Z is an oxygen atom and the cephalotaxane H—O—CTX is a cephalotaxine of the following formula, or a salt thereof:

[0239] where R¹, R², R³ and R⁴ are independently hydrogen, hydroxyl group or alkoxide.

[0240] A cephalotaxane H—O—CTX, as defined above, is cephalotaxine, or a salt thereof, where R¹ is hydroxyl, R² is methoxyl, R³ and R⁴ are hydrogen.

[0241] R⁵ is preferably hydrogen or —CH₂—CO—O—Me.

[0242] The W—CO radical is preferably such as n=1 to 4, R⁶ and R⁸ are methyl.

[0243] The W—CO radical may be too such as n=1 or 2, R⁶ is phenyl and R⁸ is hydrogen.

[0244] When R⁵ is —CH₂—CO—O—Me, R¹=OH, R²=OMe, R³=R⁴=H, the cephalotaxane is preferably such as n=0, Z is a nitrogen atom and is hydrogen.

[0245] A may be W—CO—CO where W is defined as above, or an halide.

[0246] A may also be a radical of compound W—CO—A having the ability to generate cleavage of the bond between carbonyl group and substituent A to provide W—CO⁺ and A⁻.

[0247] In addition, A is a radical selected from sub stituents:

[0248] méthoxyformyloxy of formula MeOCOO—,

[0249] trifluoroacetyloxy of formula CF₃COO—,

[0250] alkylsulfonoxy of formula RSO₃—,

[0251] phosphoxy of formula (RO)₂PO—,

[0252] halophosphoxy of formula ROP(Cl)O—,

[0253] trialkylsilyloxy of formula R₃SiO—,

[0254] formulas wherein R is alkyl,

[0255] diméthyl-formamidinium chloride of formula

[0256] or acyloxy-pyridinium bromide of formula

[0257] A may also be 2,4,6-trichlorobenzoyloxy or a radical corresponding to the following formula:

[0258] In the case where A is a carbonyl-diimidazole, where A is 2,4,6-trichlorobenzoyloxy, the reagent of formula W—CO—A is obtained by contacting an acid W—CO—OH, as defined above, with 2,4,6-trichlorobenzoyloxy carbonyl-diimidazole in presence of a strong base such as an alkoxide.

[0259] According carbodiimide method, the coupling additive is a substituted carbodiimide and/or a basic additive such as tertiary amine for example. For example, the substituted carbodiimide is selected from cyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC) and chlorhydrate of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.

[0260] The cephalotaxine alcoxide, corresponding to the formula M—O—CTX where M and CTX are as defined above, may be obtained by contacting a cephalotaxine of formula H—O—CTX with metal himself, an amidure, a metallic hydride or an alkyl-metal.

[0261] M may be an alkaline metal such as lithium, potassium or sodium.

[0262] The aim of the present invention is also the preparation of new compounds such as:

[0263] the lithium alcoxide of cephalotaxine corresponding to the following formula:

[0264] the sodium alcoxide of cephalotaxine corresponding to the following formula:

[0265] a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

[0266] where

[0267] n is included between 0 and 8;

[0268] Z is oxygen, nitrogen or sulfur heteroatom;

[0269] R⁵, R⁶ and R⁸ are independently

[0270] hydrogen;

[0271] hydrocarbon radical, saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl, of said radical including or not heteroatom(s);

[0272] oxygen ether bearing one of the former radicals;

[0273] CTX is as defined above;

[0274] except for compounds where Z is oxygen atom and,

[0275] 1°) n=2 or 3, and simultaneously R⁶=R⁸=methyl and R⁵=—CH₂COOMe,

[0276] 2°) n=2 and simultaneously R⁶=R⁸=methyl and R⁵=OMe or hydroxyl;

[0277] 3°) n=3 and simultaneously R⁶ is hydroxyl, when R⁸ is méthyl and R⁵ is —CH₂CO₂CH₃ radical.

[0278] a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

[0279] where

[0280] m, R⁵, R⁶, R⁸ and CTX are as defined above;

[0281] except compound where m=2, R⁵=CH₂CO₂CH₃, R⁶=R⁸=methyl and CTX is as defined above.

[0282] R⁵ is preferably the —CH₂—CO—O—CH₃ radical.

[0283] a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

[0284] where n, Z, Q¹, Q², R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹ and CTX are as defined above.

[0285] Preferably, Q² is oxygen atom and/or Z is nitrogen atom and the cephalotaxane such as n=0.

[0286] a sidechain-bearing cephalotaxane corresponding to the following formula:

[0287] a sidechain-bearing cephalotaxane corresponding to the following formula:

[0288] a sidechain-bearing cephalotaxane corresponding to the following formula:

[0289] When the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt

[0290] thereof, presents the following formula:

[0291] where n, R⁵, R⁶, R⁸, CTX and Z are as defined above, the said chain is open with an agent and/or a protonic or not protonic electrophilic radical E in aqueous or not aqueous medium, to provide an intermediate compound of the following formula:

[0292] where n, CTX, R⁵, R⁶ and R⁸ are as defined above, E is either hydrogen or the provisionally or definitively fixed eletrophilic radical;

[0293] the aforementioned intermediate compound may be attacked with an agent or a nucleophilic radical Z′, deliberately added or possibly present in the medium, and

[0294] when the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt

[0295] thereof, presents the following formula:

[0296] where n, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined above, and Z′ is an heteroatom;

[0297] the said chain is open by hydrolysis or carefully solvolysis with possibly presence of activation and/or opening additive.

[0298] In addition, to provide an open sidechain-bearing cephalotaxane of the following formula:

[0299] where n, CTX, R⁵, R⁶ and R⁸ are as defined above; Z′ is:

[0300] either a halogen or an heteroatom bearing a hydrogen or a radical R¹¹ such as defined above;

[0301] or an hydrogen, hydrocarbon radical, the said radical bearing or not heteroatom(s), saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, or heterocycloalkyl.

[0302] For example, cephalotaxine esters of the following formulas:

[0303] Where R⁵, R⁶, R⁸, Z′, X and CTX are as defined above;

[0304] bromodeoxyharrintonine (n=2) and bromodeoxyhomoharrintonine (n=3)

[0305] where CTX is as defined above;

[0306] aminodeoxyharrintonine (n=2) and aminodeoxyhomoharrintonine (n=3)

[0307] where CTX is as defined above;

[0308] In addition, when the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, presents the following formula:

[0309] where n, R⁵, R⁶, R⁸, CTX and Z are as defined above,

[0310] the said chain is open by treatment with a solution of hydrobromic acid in acetic acid, in an halogenated solvent, preferably dichloromethane, followed by in situ hydrolysis to provide, without isolation of the intermediate, a sidechain-bearing cephalotaxane of the following formula:

[0311] where n, CTX, R⁵, R⁶ et R⁸ are defined above.

[0312] Acids corresponding to the following formula:

Ω-CO—OH

[0313] where Ω radical is as defined according above;

[0314] the said formula equivalent to racemic mixture containing compounds of the formulas (+)-Q-CO—OH and (−)-Q-CO—OH such as (+)-Q-CO—OH represents its dextrogyre enantiomer and (−)-Q-CO—OH represent its levogyre enantiomer, were obtained

[0315] a) by contacting of said racemic mixture or one of its activated form of the formula

Ω-CO—A

[0316] which is as defined above;

[0317] the said racemic mixture or said activated form generating respectively:

[0318] either an anion corresponding to the formula (Ω-CO—O)⁻;

[0319] or a cation corresponding to the formula (Ω-CO)⁺;

[0320] with a pure enantiomeric form of chiral entity, said “resolution agent” symbolized by Δ* (delta stella), having the ability to form:

[0321] either a stable combination, by covalent bonding;

[0322] or an easily reversible labil combination, by hydrogen bonding or by hydrophobic interaction;

[0323] or intermediate lability bonding by electrostatic interaction;

[0324] to provide a diastereomeric mixture of Ω-CO—O—A* and de Ω-CO-Δ*;

[0325] b) then by physical separation of the mixture of two diastereomers or two complex compounds or more generally of two new entities physically and/or chemically different then obtained;

[0326] c) then by regeneration and finally separation of each one of enantiomers of the generic formula Ω*-CO—OH, where Ω* (<<oméga stella>>) represents the generic symbol of the same chiral radical in the either one or the other pure enantiomeric forms corresponding to the following formulas (+)-Q-CO—OH and (−)-Q-CO—OH which are as defined above.

[0327] Preferably, Ω-CO— is:

[0328] either a radical corresponding to the following formula:

[0329] where n, Z, R⁶, R⁸, and R⁵ are as defined above;

[0330] or a radical corresponding to the following formula:

[0331] where m, Z, R⁶, R⁸, and R⁵ are as defined above;

[0332] or a radical corresponding to the following formula:

[0333] where n, R⁵, R⁶ R⁸, Z, Q², Q¹, R⁹, R¹⁰ and R¹¹ are as defined above.

[0334] The stable combination may be represented by an ester of the following formula Ω-CO—O-Δ* such as Ω and Δ* are as defined above, the said stable combination is obtained by contacting acid with a chiral alcohol corresponding to the formula HO-Δ* such as Δ* is as defined above, according the process of invention.

[0335] The stable combination may be represented by an amide corresponding to the either one or the other formulas Ω-CO—NH-Δ* or Ω-CO—N-Δ* such as Ω and Δ* are as defined above, the said stable combination is obtained by contacting acid with primary or secondary chiral amine corresponding to formulas H₂N-Δ* or NN=Δ* such as Δ* is as defined above, according the process of the invention.

[0336] The stable combination may be represented by an thioester of the following formula Ω-CO—S-Δ* such as Ω and Δ* are as defined above, the said stable combination is obtained by contacting acid with a chiral thiol corresponding to the formula HS-Δ* such as Δ* is as defined above, according the process invention.

[0337] Finally, the ionic combination may be represented by a salt just prepared by contacting of acid with a chiral amine corresponding to the either one or the other of the three following formulas:

Ω-CO—O⁻ [NH-Δ*]⁺

Ω-CO—O⁻ [NH₂-Δ*]⁺

Ω-CO—O⁻ [NH₃-Δ*]⁺

[0338] where Ω and Δ* are as defined above.

[0339] The bringing into play of a labile bonding based combination is achieved in the form of chromatography with the help of a chiral stationary phase.

[0340] The bringing into play of an interatomic or intermolecular labile bonding based combination, within crystalline latice, is achieved in the form of fractionated crystallization initiated by a chiral precursor.

[0341] The chiral alcohol HO-Δ* is:

[0342] either (−)-quinine corresponding to the following formula:

[0343] or (−)- or (+)-methyl mandelate corresponding to the following formulas:

[0344] or or (+)-menthol corresponding to the following formulas:

[0345] The chiral amine H₂N-Δ* is (−)- or (+)-ephedrine corresponding to the following formulas:

[0346] The present invention concerns the following new compounds:

[0347] the (−)-quinidyl (2′R)-(−)-anhydro-homoharringtonate and the (−)-quinidyl (2′S)-(−)-anhydro-homoharringtonate corresponding respectively to the two following formulas:

[0348] the (−)-menthyl (2′R)-(−)-anhydro-homoharringtonate and the (−)-menthyl (2′S)-(−)-anhydro-homoharringtonate corresponding respectively to the two following formulas:

[0349] the (−)-methylmandelyl (2′R)-(−)-anhydro-homoharringtonate and the (−)-methylmandelyl (2′S)-(−)-anhydro-homoharringtonate corresponding respectively to the two following formulas:

[0350] the (−)-ephedrinium (2′R)-(−)-anhydro-homoharringtonate and the (−)-ephedrinium (2′S)-(−)-anhydro-homoharringtonate corresponding respectively to the two following formulas:

[0351] According the process of invention, when the carboxylic acid is the tertiary heterocycloalcane carboxylic acid corresponding to the following formula:

[0352] where n, Z, R⁵, R⁶ and R⁸ are as defined above, the said acid is obtained by treatment in aprotic or protic solvant, eventually in the presence of cyclization additive and/or dehydrating agent, the said treatment eventually supported with physical carrying of the water formed.

[0353] or open tertiary ethylenic acid corresponding to the following formula:

[0354] where m, Z, R⁵, R⁶ and R⁸ are as defined above.

[0355] or open tertiary ethylenic acid corresponding to the following formula:

[0356] where m is included between 1 and 8, Z, R⁵, R⁶ and R⁸ are as defined above, R¹² is not a CTX radical as defined above and represents R⁵ and/or a protective group of acids and/or a chiral group;

[0357] then R¹² is removed later, either just by saponification, or by hydrogenolysis, or more generally by the method of the state of art to remove protective groups of acids.

[0358] In the absence of cyclization additive, the reaction of cyclization just take place by heating.

[0359] Preferably, the cyclization additive is a protic acid such as sulfonic or formic acid, or an aprotic acid, included in immobilized form.

[0360] In the step of preparation of the acid described above, Z is an oxygen atom.

[0361] A cephalotaxane H—O—CTX of the following formula, or a salt thereof, or a diastereoisomer thereof:

[0362] where R¹³ and R¹⁴ are independently hydrogen, hydroxyl or alkoxide and R¹⁵ to R¹⁸ and R²⁰ to R²⁶ are independently hydrogen, hydroxyl, thiol, halogen, alcoxy, alkylthio group, or alkoxide, or R¹⁵, R¹⁷ and R¹⁸ can further independently represent OR¹⁹, R¹⁹ being hydrogen or an hydrocarbon radical.

[0363] R²⁰ to R²⁶ may be hydrogen, and R¹³ and R¹⁴ may be independently hydroxyl or hydrogen when R¹⁵ to R¹⁸ are defined as above.

[0364] R¹⁶ and R²⁰ to R²⁶ may be hydrogen, R¹³ may be hydroxyl, R¹⁴ may be hydrogen, and R¹⁵, R¹⁷ and R¹⁸ may be independently OR¹⁹, R¹⁹ being alkyl, alkenyl, alkynyl, cycloalkyl, cycloaklenyl, aryl or heteroalkyl, said radicals including or not heteroatom(s).

[0365] A cephalotaxane H—O—CTX, as defined above, where the H—O—CTX is drupacine of formula:

[0366] or a salt thereof.

[0367] R¹ and R⁴ are hydroxyl, R² is hydrogen and R³ may be methoxyl, wherein said cephalotaxane H—O—CTX is 11-hydroxy-cephalotaxine, or a salt thereof, of formula:

[0368] A sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

[0369] where

[0370] n is included between 0 and 8;

[0371] Z is oxygen, NH or sulfur heteroatom;

[0372] R⁵, R⁶ and R⁸ are independently hydrogen, hydrocarbon radical, saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl, of said radical including or not heteroatom(s), oxygen ether bearing one of the former radicals;

[0373] CTX is as defined as above.

[0374] R⁵ may be CH₂CO₂Me and R⁶=R⁸=methyl, corresponding to the following formula and/or a salt thereof:

[0375] CTX may be a drupacine radical.

[0376] CTX may be a 1 1-hydroxy-caphalotaxine radical.

[0377] The present invention also concerns a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

[0378] where n, CTX, R⁵, R⁶ and R⁸ are as defined as above and Z′ is defined as above;

[0379] except for compounds where Z′ is hydrogen and simultaneously R⁶=R⁸=methyl and R⁵ is CH₂CO₂Me and,

[0380] 1°) n is 2 and CTX is a drupacine radical;

[0381] 2°) n is 2 or 3, and CTX is a 11-hydroxy-cephalotaxine radical.

[0382] Z′ may be hydroxyl.

[0383] R⁵ may be CH₂CO₂Me and R⁶=R⁸=methyl and CTX is a drupacine radical corresponding to the following formula and/or a salt thereof:

[0384] The aim of the present invention is also the preparation of the following new compounds:

[0385] the tertiary heterocycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0386] where n, Z, R⁵, R⁶ and R⁸ are as defined above, and R⁵ is not hydrogen;

[0387] except for compounds where Z is oxygen atom and,

[0388] 1°) n=0 and R⁵ is not —CH₂CO₂H or —CH₂CO₂CH₃ radical;

[0389] 2°) n=0 and R⁵ is —CH₂CO₂H or —CH₂CO₂CH₃ radical, and R⁶=R⁸=methyl or —CH₂CO₂H or —CH₂CO₂CH₃ radical;

[0390] 3°) n=2 and simultaneously R⁶=R⁸=methyl, and R⁵=OMe or hydroxyl

[0391] 4°) n 32 2 and simultaneously R⁶=R⁸=methyl, and R⁵ is —CH₂CO₂H or —CH₂CO₂CH₃ radical or methyl;

[0392] 5°) n=3 and simultaneously R⁶ is hydroxyl, and R⁸ is methyl, and R⁵ is —CH₂CO₂CH₃ radical;

[0393] 6°) n=3 and simultaneously R⁶=R⁸=methyl and R⁵=OH or methyl or ethyl.

[0394] the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0395] where n is included between 0 and 8, R⁵, R⁶ and R⁸ are as defined above, but are not hydrogen simultaneously.

[0396] except for compounds corresponding to the exceptions 1 to 6 defined above.

[0397] the tertiary heterocycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0398] where n is included between 0 and 8, Z, R⁵, R⁶ and R⁸ are as defined above, R⁵ is not hydrogen, and R¹² is not a CTX radical defined above;

[0399] except for compounds where Z is oxygen atom and, corresponding to the exceptions 1 to 6 defined above.

[0400] the tertiary oxacycloalcane carboxylic hemiester, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0401] where n is included between 0 and 8, R⁶ and R⁸ are as defined above. except for compounds corresponding to the exceptions 1 to 6 defined above.

[0402] the tertiary oxacycloalcane carboxylic hemiester, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0403] where n is included between 0 and 8, R⁶ and R⁸ are as defined above, R¹² is an hydrocarbon radical different from CTX as defined above. except for compounds corresponding to the exceptions 1 to 6 defined above.

[0404] the tertiary oxacycloalcane carboxylic hemiester or anhydro-homoharringtonic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0405] the tertiary oxacycloalcane carboxylic hemiester or anhydro-harringtonic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0406] the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0407] where n and R⁵ are as defined above,

[0408] except for compounds corresponding to the exceptions 1 to 6 defined above.

[0409] the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0410] where n is included between 1 and 8.

[0411] the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0412] where n is included between 0 and 8.

[0413] the tertiary oxacycloalcane carboxylic acid or oxanhydroneoharringtonic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0414] the tertiary oxacycloalcane carboxylic acid or oxanhydro-neohomoharringtonic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0415] the tertiary oxacycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0416] the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0417] where m is included between 1 and 8, R⁶and R⁸ are as defined above, but are not hydrogen simultaneously, and R⁵ is not hydrogen or heteroatom.

[0418] the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0419] where m, R⁶, R⁸ and R¹² are as defined above, and m′ is included between 1 and 8.

[0420] the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0421] where m is included between 1 and 8, R⁶ and R⁸ are as defined above but are not hydrogen.

[0422] the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0423] the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0424] the tertiary alkene carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

[0425] where m is included between 1 to 8, preferably m 1.

[0426] the anhydrides of acid of the general formula W—CO—O—CO—W where W is as defined above.

[0427] the mixed anhydrides of acid of the general formula W—CO—A where A is a radical selected from the following substituents:

[0428] méthoxyformyloxy of formula MeOCOO—,

[0429] trifluoroacétyloxy of formula CF₃COO—,

[0430] alkylsulfonoxy of formula RSO₃—,

[0431] phosphoxy of formula (RO)₂PO—,

[0432] halophosphoxy of formula ROP(Cl)O—,

[0433] trialkylsilyloxy of formula R₃ SiO—,

[0434] formulas wherein R is alkyl.

[0435] diméthyl-formamidinium chloride of formula

[0436] acyloxy-pyridinium bromide of formula

[0437] and 2,4,6-trichlorobenzoyloxy.

[0438] the mixed anhydride corresponding to the following formula:

[0439] the acid chlorides defined above, corresponding to the general formula Ω-CO—X, where X is halogen.

[0440] the cyclic anhydrides corresponding to the following formula:

[0441] where n, R⁶ and R⁸ are as defined above.

[0442] the cyclic anhydride corresponding to the following formula:

[0443] In the process according to the invention, the sidechain-bearing cephalotaxane was purified like a salt by chromatography using a hydrophobic reversed-phase like stationary phase, and a mobile phase without organic solvent like a solution adjusted to a pH 2 to 4.5 with a buffer prepared with an acid and an alkaline or ammonium salt and one or several additive like attenuator of silanol effect, the said cephalotaxine salt was generated from mineral acid under the form of chlorohydrate, sulfate, phosphate, nitrate, perchlorate, or from organic acid under the form of tartrate, malate, citrate or lactate.

[0444] In the process according to the invention, the sidechain-bearing cephalotaxane was purified by a step of chromatographic purification of a natural or semi-synthetic or synthetic homoharringtonine as a pharmaceutical use corresponding to the following formula:

[0445] to remove the undesired related impurity named 2′-épi-homoharringtonine resulting:

[0446] a) either from a semi-synthetic process with introduction of a synthetic homoharringtonic acid of inadequate enantiomeric purity, the generated impurity showing the absolute configuration corresponding to the following formula:

[0447] b) or from the biosynthetic process in the plant, where a cephalotaxine with inadequate enantiomeric purity was introduced, or in the form of artefact by partial racemization of the cephalotaxine moiety, the generated impurity showing strictly identical chromatographic properties with a non-chiral system, with an absolute configuration opposite to the one above (enantiomer) and corresponding to the following formula:

[0448] especially making use of one of the following chromatographic systems:

[0449] A) Stationary phase: alkyl- or phenyl- or alkylphenyl- or phenylalkyl-silane, preferably n-octadecylsilane,

[0450] B) Mobile phase: water-tetrahydrofurane, water-methanol, water-acetonitrile or buffer pH 2 to 6.5 in replacement of water, or all other mobile phase with equivalent selectivity,

[0451] This process of purification and chromatographic control of a natural or semi-synthetic or synthetic homoharringtonine, allows to offset the double insufficiency of enantiomeric purity of the semi-precursors, both on the sidechain precursor (said homoharringtonic acid) and cephalotaxine, the two said-precursors are each independently generated by total synthesis or semi-synthetic process or natural process within of the plant (biosynthesis), in fact the withdrawal of the non natural enantiomer of homoharrintonine showing an opposite absolute configuration, by using a chiral stationary phase with preparative scale.

[0452] The present invention is illustrated without implied limitation by the following schemes.

[0453] Scheme 1 in annex gives the definition and the formulas of main harringtonines.

[0454] Scheme 2 recapitulates process of preparation of harringtonines of prior art.

[0455] Scheme 3 gives the sequence of synthesis of homoharringtonine corresponding to the Example 25, where A represents a 2,4,6-trichloro-phenyl group, R represents a methyl and R′ represents a cephalotaxyl moiety.

[0456] Scheme 4 represents a variant of the process according to the invention, more exactly the semi-synthesis of harringtonines via oxacycloalcalne carboxylic acids. The substitutes R⁶, R⁷, R⁸, R, A, CTX, X and the letters n et m referenced in this scheme are defined in the description.

[0457] The following definitions applies for the whole present document.

[0458] Alkaloids: natural substances present in the vegetal kingdom allowing at least a cyclic or acyclic basic nitrogen (allowed extensions: animal kingdom, primary amine; refused extensions: amidic nitrogen, because non basic, ex: taxanes) and showing frequently pronounced pharmacological properties.

[0459] Basic or free Alkaloid: alkaloid showing a tertiary amine in a non-ionized form generally existing at alkaline pH and soluble in aprotic organic solvents.

[0460] Salt of alkaloid or just <<salt>>: ionized form of alkaloid with amine function showing a positive formal charge and a negative counter-ion, actually more soluble in water and protic solvents and less soluble in aprotic solvents.

[0461] Cephalotaxanes 1 (see Scheme 1): this generic term indicates the basic framework, showing diverse oxygenated substitutes (aliphatic or aromatic ether, free or esterified alcohol, enol and/or free or substituted phenol, bridged ether, more generally all substitute usually founded at natural state for this kind of compounds).

[0462] Cephalotaxines 2: this generic term indicates cephalotaxanes possibly showing at least one of the substitutes described above, a sidechain excepted.

[0463] Cephalotaxine 2: a cephalotaxine in majority present in the genus Cephalotaxus

[0464] Cephalotaxoids: this generic term indicates a non-natural cephalotaxine.

[0465] Harringtonines: this generic term indicates cephalotaxane showing at least an alcohol group, a phenol or an enol, esterified by a sidechain and possibly by one of the substitutes described above.

[0466] Harringtonine (the): one of the main alkaloids bearing a sidechain in position 3. Harringtoids: this generic term indicates a non-natural harringtonine, where sidechain is an ester radical showing at least 3 carbon atoms.

[0467] Sidechain of the harringtoids: this generic term indicates an ester formed between one of the hydroxyl group and a carboxylic acid showing at least 3 carbon atoms allowing usually a tertiary alcohol tertiaire in a position and an hydrophobe substitute in w position relatively to the carboxyl group.

[0468] Adaptation of the additive et subtractive empirical nomenclature suitable for cephalotaxanes.

[0469] Prefixes of common nomenclature are usually used in the literature to indicate structural variations of the sidechain of harringtonines (see Examples in the table p. 134). The sidechain of reference is this of harringtonine showed in formula 3b, n=2, R=H, R⁷=H, R⁶=2-hydroxyisopropyl.

[0470] Homo: 1 extra carbon.

[0471] Bishomo: 2 extra carbons.

[0472] Nor: a sidechain with 1 carbon less.

[0473] Iso: a sidechain with methylene bearing an hydroxyl group at the place of juxtaterminal carbon.

[0474] Deoxy: the hydroxyl group of juxtaterminal carbon is replaced par hydrogen.

[0475] Anhydro: the two tertiary hydroxyl groups lose a molecule of water to give the corresponding saturated oxygenated heterocycle.

[0476] neo: R⁶ is a phenyl group at the place of 2-hydroxyisopropyl.

[0477] HPLC: High-Performance Liquid Chromatography.

[0478] NMR: Nuclear Magnetic Resonance.

[0479] The following examples, which are given without implied limitation, illustrates the present invention.

EXAMPLE 1

[0480] Preparation of Ethyl 2-methoxycarbonylmethyl-2-hydroxy-6-methylhept-5-enoate or Ethyl 6-desoxy-5,6-dehydrohomoharringtonate:

[0481] 1°) Preparation of the Intermediate Oxalate

[0482] 5-Bromo-2-methyl-pent-2-ene (15.6 g, 95.6 mmol) was added dropwise to a stirred mixture of magnesium (2.32 g, 95.5 mmol)(activated with further crystal of iodine) in anhydrous tetrahydrofurane (75 ml). The onset of the reaction is accompanied with a vigorous overheating and refluxing of the reaction mixture. The reflux was maintained until most of magnesium had reacted and the reaction mixture was diluted with anhydrous tetrahydrofurane (150 ml). To a stirred mixture of diethyl oxalate (10.8 ml, 80 mmol) in anhydrous tetrahydrofurane (75 ml) was added at −78° C. the resulting Grignard reagent over a period of 20 minutes. The stirring was maintained at −78° C.±5° C. for 30 minutes and then the temperature was raised to −10° C. over a period of 1.5 hours. The mixture was quenched with 15% ammonium chloride solution (300 ml). The separated organic layer was washed with 15% ammonium chloride solution (300 ml) and evaporated to dryness. The aqueous layer was extracted with ether (2×300 ml). The organic layers were combined with the concentrate and washed with brine (300 ml), dried over magnesium sulfate and evaporated to dryness. The crude product, after purification with a bulb-to-bulb distillation apparatus, afforded colorless oil (10.3 g, 70%). The intermediate a-cetoester showed the following characteristics:

[0483] IR (ATR) (cm⁻¹): 2790; 2916; 1725; 1068.

[0484]¹H NMR 400 MHz (CDCl₃)(d ppm, J Hz): 5.08 (1H, t, J=7.2, HC═); 4.32 (2H, q, J=7.1, OCH₂); 2.86 (2H, t, J=7.2, CH₂CO); 2.32 (2H, q, J=7.2, CH₂—C═); 1.68 (3H, s, CH₃); 1.62 (3H, s, CH₃); 1.37 (3H, t, J=7.1, OCH₂CH₃).

[0485] 2°) Preparation of the Title Product

[0486] Anhydrous methyl acetate (0.6 ml, 7.5 mmol) was added to a stirred commercial solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane (7.5 ml, 7.5 mmol) at −78° C. over a period of 1 minute and this was allowed the reaction to proceed at −78±5° C. for 20 minutes. To a stirred mixture of ethyl 2-oxo-6-methylhept-5-enoate prepared above (480 mg, 2.6 mmol) in anhydrous tetrahydrofurane (10 ml) at −78° C. was added the lithium enolate over a period of 5 minutes, and the resulting mixture was stirred at −78±5° C. for 30 minutes. After monitoring in CCM, the freezing bath was removed and the mixture was quenched with 15% ammonium chloride solution (10 ml). The separated organic layer was washed with 15% ammonium chloride solution (10 ml) and evaporated to dryness. The aqueous layers were extracted with ether (2×10 ml). The organic layers were combined with the concentrate and washed with brine (10 ml), dried over magnesium sulfate and evaporated to dryness. The resulting crude product (1.13 g) was purified by column chromatography (cyclohexane/ethyl acetate (95:5), silica (15-40 mm) 38 g) to provide a colorless oil (482 mg, 72%). The product thus obtained showed the following characteristics:

[0487] IR (ATR) (cm⁻¹): 3508; 2969; 2919; 1732; 1438; 1193.

[0488]¹H NMR 400 MHz (CDCl₃)(d ppm, J Hz): 5.05 (1H, t, J=7.1, HC═); 4.27 (2H, q, J=7.1, OCH₂); 3.70 (1H, s, OH); 3.68 (3H, s, OCH₃); 2.92 and 2.70 (2H, 2d, J_(AB)=16.1, CH₂CO₂); 2.12 (1H, m); 1.88 (1H, m); 1.72 (2H, m); 1.67 (3H, s, CH₃); 1.58 (3H, s, CH₃); 1.31 (3H, t, J=7.1, OCH₂CH₃).

EXAMPLE 2

[0489] Preparation of ethyl 2-methoxycarbonylmethyl-2-hydroxy-6,6-dimethyl-2-tetrahydropyrane carboxylate of ethyl anhydrohomoharringtonate:

[0490] 1°) Method A

[0491] p-Toluenesulfonic acid (2,06 g, 10,8 mmol) was added to a stirred solution of ethylenic ester resulting from Example 1 (2.8 g, 10,8 mmol) in toluene (30 ml) and the resulting mixture was stirred at 65° C. for 5 hours. After cooling at room temperature, the mixture was hydrolyzed with saturated sodium hydrogen carbonate solution. The aqueous layer was extracted with ether (3×50 ml), and the organic layers were combined, washed with brine (100 ml), dried over magnesium sulfate and evaporated to dryness. The resulting crude product (2.8 g) was purified by column chromatography (cyclohexane/ether (95:5), silica (15-40 mm) 110 g) to provide a colorless oil (1.94 g, 69%). The product thus obtained showed the following characteristics:

[0492]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 4.21 (2H, m, OCH₂CH₃); 3.64 (3H, S, OCH₃); 2.85 and 2.60 (2H, 2d, J_(AB)=14.0, CH₂CO₂); 2.30 (1H, dt, J=13.3 and 3.7); 1.87 (1H, qt, J=13.8 and 3.6); 1.62 (1H, m); 1.51 (2H, m); 1.43 (1H, m); 1.31 (3H, t, J=7.1, OCH₂CH₃); 1.22 (3H, s, CH₃); 1.13 (3H, s, CH₃).

[0493] 2°) Method B

[0494] To a stirred solution of ethylenic ester resulting from Example 1 (50 mg, 0.19 mmol) in methanol (30 ml) was added hydrochloric acid 1N (0.5 ml) and the resulting mixture was stirred at 65° C. for 15 hours. After dilution with dichloromethane, the organic layer was dried over magnesium sulfate and evaporated to dryness. The resulting crude product (32 mg) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (9:1), silica (15-40 mm) 2.2 g) to provide the expected intermediate diol (20 m g, 37%). The product thus obtained showed the following characteristics:

[0495] IR (ATR) (cm⁻¹): 3490; 2966; 1731; 1193; 1177; 1152.

[0496]¹H NMR 400 MHz (CDCl₃)(d ppm, J Hz): 4.28 (2H, q, J=7.2, OCH₂); 3.75 (1H, s, OH); 3.68 (3H, s, OCH₃); 2.93 and 2.69 (2H, 2d, J_(AB)=16.2, CH₂CO₂); 1.70 (2H, m); 1.53 (1H, m); 1.44 (1H, m); 1.30 (3H, t, J=7.1, OCH₂CH₃); 1.20 (3H, s, CH₃); 1.19 (3H, s, CH₃).

[0497] To a stirred solution of diol prepared above (19 mg, 0.069 mmol) in 1,2-dichloroethane (1.4 ml) was added anhydrous zinc chloride (10 mg, 0.069 mmol) and the resulting mixture was stirred at 80° C. for 1.5 hours. After cooling at ambient temperature, the mixture was washed with water, then with brine, and the aqueous layer was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford cyclic diester (7 mg, 40%). The product thus obtained showed identical characteristics to this obtained with method A.

[0498] 3°) Method C

[0499] A solution of ethylenic ester resulting from Example 1 (400 m g, 1.55 mmol) in a mixture of formic acid (4 ml) and water (4 ml) was stirred at 50° C. for 15 hours. After removal of formic acid in vacuo, the residue was treated with 5% sodium hydrogen carbonate solution. The aqueous layer was extracted three times with dichloromethane then the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (375 mg) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (98:2), silica (15-40 mm) 16 g) to provide a colorless oil (235 mg, 55%). The product thus obtained showed identical characteristics to this obtained with method A. The cyclisation of the diol thus obtained with zinc chloride, like Example 2 method B above, afforded cyclic diester showing identical characteristics to this obtained with method A.

EXAMPLE 3

[0500] Preparation of 2-carboxymethyl-2-hydroxy-6-methylhept-5-enoic acid or O-demethyl-6-desoxy-5,6-dehydrohomoharringtonic acid:

[0501] A mixture of potassium hydroxide (14.2 g, 252 mmol) in water (170 ml) was added to a stirred solution of ethylenic ester resulting from Example 1 (10.95 g, 42 mmol) in methanol (300 ml) and the resulting mixture was stirred at reflux for 1.5 hours. After cooling at room temperature, and removal of methanol in vacuo, the residue was treated with water (10 ml) and the resulting aqueous layer was extracted with ether (250 ml). After acidification (pH 1) with 10% hydrochloric acid, the aqueous layer was extracted with ether (3′ 250 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a white solid (8.66 g, 95%). The crude product thus obtained showed the following characteristics:

[0502] IR (ATR) (cm⁻¹): 3500; 3019; 2966; 2931; 1716; 1691; 1656; 1219; 1199; 1111.

[0503]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 5.06 (1H, t, J=6.9, HC═) 3.04 and 2.78 (2H, 2d, J_(AB)=17.1, CH₂CO₂); 2.25-1.20 (4H, m, 2×CH₂); 1.67 (3H, s, CH₃); 1.60 (3H, s, CH₃).

EXAMPLE 4

[0504] Preparation of 2-carboxymethyl-6,6-dimethyl-2-tetrahydro-pyranecarboxylic acid or O-demethylanhydrohomoharringtonic acid:

[0505] 1°) Method A

[0506] A mixture of potassium hydroxide (4.2 g, 75 mmol) in water (45 ml) was added to a stirred solution of cyclic diester resulting from Example 2 (1.94 g, 7.5 mmol) in ethanol (75 ml) and the resulting mixture was stirred at reflux for 5 hours. After cooling at room temperature, and removal of ethanol in vacuo, the residue was treated with water (10 ml) and the resulting aqueous layer was extracted with ether (2′ 250 ml). After acidification with hydrochloric acid 2N (35 ml), the aqueous layer was saturated with sodium chloride then was extracted with ether (3′ 50 ml). The combined organic layers were washed with brine (2′ 100 ml) dried over magnesium sulfate and evaporated to dryness to afford a pale yellow oil (1.66 g, 98%). The crude product thus obtained showed the following characteristics:

[0507] IR (ATR) (cm⁻¹): 2974; 2941; 1709; 1215.

[0508]¹H NMR 400 MHz (CDCl₃)(d ppm, J Hz): 3.01 and 2.95 (2H, 2d, J_(AB)=16.1, CH₂CO₂); 1.89 (1H, m); 1.75 (2H, m, CH₂); 1.58 (3H, m); 1.31 (6H, s, 2×CH₃).

[0509] 2°) Method B

[0510] To a stirred solution of ethylenic diacid resulting from Example 3 (50 mg, 23 mmol) in anhydrous toluene (500 ml) was added zinc chloride (6 mg, 0.04 mmol) and the resulting mixture was stirred at 80° C. for 15 hours. After cooling at room temperature, the mixture was hydrolyzed with 10% hydrochloric acid, and the resulting aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a pale yellow solid (38 mg, 76%). The crude product thus obtained showed identical characteristics to this obtained with method A.

[0511] 3°) Method C

[0512] A solution of ethylenic diacid resulting from Example 3 (50 m g, 0.23 mmol) in a mixture of formic acid (500 ml) and water (500 ml) was stirred at 60° C. for 3 hours. After cooling at room temperature and removal of formic acid in vacuo, the residue was treated with ethyl acetate. The resulting organic layer was washed with 10% hydrochloric acid and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a pale yellow solid (50 mg, 100%). The crude product thus obtained showed identical characteristics to this obtained with method A .

EXAMPLE 5

[0513] Preparation of 2-methoxycarbonylmethyl-2-hydroxy-6-methylhept-5-enoic acid or 6-desoxy-5,6-dehydrohomoharringtonic acid:

[0514] A mixture of ethylenic diacid resulting from Example 3 (500 mg, 2.3 mmol) and a commercial solution of boron trifluoride-methanol complex in methanol (4.5 ml, BF₃ 12% w/w) was stirred at 18±5° C. for 16 hours. After careful addition of the reaction mixture at saturated sodium hydrogen carbonate solution (50 ml), the resulting aqueous layer was washed with ether (50 ml), acidified (pH 1) with hydrochloric acid 2N (0.5 ml and extracted with ether (3′ 50 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a viscous yellow oil (310 mg, 58%). The crude product thus obtained showed the following characteristics:

[0515] IR (ATR) (cm⁻¹): 3483; 2954; 1731; 1197; 1173.

[0516]¹H NMR 400 MHz (CDCl₃)(d ppm, J Hz): 5.06 (1H, m, HC═); 4.12 (2H, br.s, CO₂H+OH); 3.73 (3H, s, OCH₃); 2.99 and 2.74 (2H, 2d, J_(AB)=16.7, CH₂CO₂); 2.16 (1H, m); 1.98 (1H, m); 1.85-1.60 (4H, m); 1.67 (3H, s, CH₃); 1.60 (3H, s, CH₃).

EXAMPLE 6

[0517] Preparation of 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydropyranecarboxylic acid or anhydrohomoharringtonic acid:

[0518] 1°) Preparation from Cyclic Diacid

[0519] A mixture of cyclic diacid resulting from Example 4 (1.6 mg, 7.4 mmol) and a commercial solution of boron trifluoride-methanol complex in methanol (15.5 ml, BF₃ 12% w/w) was stirred at 18±5° C. for 15 hours. After careful addition of the reaction mixture at saturated sodium hydrogen carbonate solution (50 ml), the resulting aqueous layer was washed with ether (2×50 ml) (to see annex preparation below), acidified (pH 1) with hydrochloric acid 2N (15 ml) and extracted with ether (3′ 75 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a yellow oil (1.17 g, 69%). The crude product thus obtained showed the following characteristics:

[0520] IR (ATR) (cm⁻¹): 2974; 2951; 1740; 1718; 1437.

[0521]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 3.70 (3H, s, OCH₃); 3.03 and 2.98 (2H, 2d, J_(AB)=16.1, CH₂CO₂); 1.82 (1H, m); 1.74 (3H, m); 1.62 (1H, m); 1.48 (1H, m); 1.31 (3H, s, CH₃); 1.26 (3H, s, CH₃).

[0522] Annex Preparations:

[0523] a) Obtaining of Diester

[0524] The combined organic layers above-mentioned was dried over magnesium sulfate and evaporated to dryness to afford a mixture of diester and monoester (396 mg). After treatment of this mixture with saturated sodium hydrogen carbonate solution, the aqueous layer was extracted with ether,and the resulting organic layer was dried over magnesium sulfate and evaporated to dryness to afford an oil (292 mg, 17%). The crude product thus obtained showed the following characteristics:

[0525]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 3.75 (3H, s, OCH₃); 3.65 (3H, s, OCH₃); 2.85 and 2.61 (2H, 2d, J_(AB)=14.1, CH₂CO₂); 1.85 (1H, m); 1.62 (1H, m); 1.50 (2H, m); 1.43 (1H, m); 1.21 (3H, s, CH₃); 1.11 (3H, s, CH₃).

[0526] b) Obtaining of Regio-hemiester by Mono Saponification of Diester Above-mentioned

[0527] To a stirred solution of cyclic diester above-mentioned (285mg, 1.17 mmol) in methanol (11 ml) was added a mixture of potassium hydroxide (654 mg, 11. 7 mmol) in water (7 ml) and the resulting mixture was stirred at room temperature for 30 minutes. After removal of methanol in vacuo, the residue was treated with water (7 ml) and the resulting aqueous layer was extracted three times with ether.

[0528] After acidification (pH 1) with 10% hydrochloric acid solution, the aqueous layer was extracted with. The combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (236 mg) was purified by column chromatography (dichloromethane/methanol (95:5), silica (15-40 mm) 6.5 g) to provide a pale yellow solid (220 mg, 82%). The product thus obtained showed the following characteristics:

[0529] IR (KBR) (cm⁻¹): 3421; 2960; 2929; 1744; 1705; 1209.

[0530]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 3.76 (3H, s, OCH₃); 2.76 and 2.67 (2H, 2d, J_(AB)=15.3, CH₂CO₂); 2.36 (1H, m, J_(AB)=13.7, J₃₋₄=3.5, J₃₋₅=1.2, H-³ _(eq)); 1.85 (1H, m, J_(AB)˜J_(ax-ax)=14.0, J_(ax-eq)=3.7, H-4_(ax)); 1.67 (1H, m, J_(AB)=14.1, J_(4-3,5)=3.9, H-4_(eq)); 1.59 (1H, m, J_(AB)=13.4, J₅₋₄=3.6, J₅₋₃=1.0, H-5_(eq)); 1.49 (1H, m, J_(AB)˜J_(ax-ax)=13.2, J_(ax-eq)=4.0, H-3_(ax)); 1.42 (1H, m, J_(AB)˜J_(ax-ax)=13.2, J_(ax-eq)=4.5, H-5_(ax)); 1.33 (3H, s, CH₃); 1.16 (3H, s, CH₃).

[0531] 2°) Preparation from Ethylenic Hemiester

[0532] To a stirred solution of ethylenic hemiester resulting from Example 5 (4.6 g, 20 mmol) in toluene (125 ml) was added p-toluenesulfonic acid (3.8 g, 20 mmol) and the resulting mixture was stirred at 65° C. for 5 hours. After cooling at room temperature, the mixture was hydrolyzed with saturated sodium hydrogen carbonate solution (100 ml). The aqueous layer was washed with ether (2×100 ml) and the organic layers were discarded (to eliminate resulting diester of the reaction). After acidification (pH 1) with hydrochloric acid 1N (35 ml), the aqueous layer was saturated with sodium chloride then was extracted with ether (3′ 100 ml). The combined organic layers were washed with brine (100 ml) dried over magnesium sulfate and evaporated to dryness.

[0533] The resulting crude product (3.9 g) was purified by column chromatography (dichloromethane/methanol (99:1), silica (15-40 mm) 160 g) to provide a yellow oil (3.1 g, 67%). The crude product thus obtained showed identical characteristics to this obtained above.

EXAMPLE 7

[0534] Preparation of cyclic anhydride of 2-carboxymethyl-2-hydroxy-6-methylhept-5-enoic acid

[0535] A mixture of cyclic diacid resulting from Example 4 (245 mg, 1.1 mmol) and acetic anhydride (4 ml) was stirred at reflux for 16 hours. After evaporation of reaction mixture in vacuo, the residue was treated with toluene and evaporated again in high vacuum to afforde an viscous yellow oil (189 mg, 84%) The product thus obtained showed the following characteristics:

[0536] IR (ATR) (cm⁻¹): 2976; 2951; 1732; 1188; 1170

[0537]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 3.02 (2H, s, CH₂CO₂); 1.98 (2H, m, CH2); 1.8-1.5 (4H, m, CH₂); 1.31 (3H, s, CH₃); 1.22 (3H, s, CH₃).

EXAMPLE 8

[0538] Preparation of (−)-cephalotaxyl pivalate:

[0539] 1 °) Method via Mixed Anhydride

[0540] To a stirred mixture of pivalic acid (100 mg, 0.98 mmol) in anhydrous toluene (2 ml) was added at room temperature triethylamine (dried over potassium hydroxide) (138 ml, 0.98 mmol) and 2,4,6-trichlorobenzoyl chloride (153 ml, 0.98 mmol). After stirring at 18±5° C. for 1.5 hours (with control of disappearing of starting acid in infra-red), 4-dimethylaminopyridine (139 mg, 1. 14 mmol) was added the reaction mixture was allowed to react for 5 minutes and cephalotaxine (103 mg, 0.33 mmol) was added. After stirring at 18±5° C. for 15 hours, the reaction mixture was filtered on paper and diluted with ether (5 ml). The resulting organic layer was successively washed with water (5 ml), with saturated sodium hydrogen carbonate solution (5 ml), with water again (5 ml) then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 mm)) to provide a solid (130 mg, 93%). The product thus obtained showed the following characteristics:

[0541]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 6.60 (1H, s, H-17*); 6.58 (1H, s, H-14*); 5.84 et 5.83 (2H, 2d, J_(AB)=1.5, OCH₂O); 5.83 (1H, d, H-3); 5.02 (1H, s, H-1); 3.77 (1H, d, J₄₋₃=9.6, H-4); 3.69 (3H, s, OCH₃); 3.21 (1H, m, J_(AB)=14.0, J=12.5, 7.8, H-11b); 3.09 (1H, m, H-8a); 2.94 (1H, td, J=11.5, 7.1, H-10a); 2.57 (2H, m, H-8b +H-10b); 2.35 (1H, dd, J_(AB)=14.5, J=6.9, H-11a); 2.03 (1H, td, J_(AB)=12.1, J=9.7, H-6_(A)); 1.89 (1H, m, J_(AB)=12.1, J=7.9, 4.0, H-6_(B)); 1.75 (2H, m, CH₂-7); 0.83 (9H, s, C(CH₃)₃).

[0542] 2°) Method Using DCC

[0543] To a stirred mixture of pivalic acid (50 mg, 0.49 mmol) in anhydrous toluene (2 ml) maintained in an inert atmosphere was added 1,3-dicyclohexylcarbodiimide (130 mg, 0.63 mmol). After stirring for 10 minutes at room temperature, cephalotaxine (50 mg, 0.16 mmol) and pyrrolidinopyridine (24 mg, 0.16 mmol) were added. After stirring at 18±5° C. for 2 hours, then at 50° C. for 15 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product (130 mg) was purified by column chromatography (dichloromethane/methanol (9:1), silica (15-40 mm) 3g) to provide a white solid (36 mg, 57%). The crude product thus obtained showed identical characteristics to this obtained above via mixed anhydride.

EXAMPLE 9

[0544] Preparation of (−)-cephalotaxyl 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydropyrane carboxylate or anhydrohomoharringtonine and methyl 2-cephalotaxyloxy-carbonylmethyl-6,6-dimethyl-2-tetrahydropyrane carboxylate, from cyclic anhydride resulting from Example 7:

[0545] To a stirred mixture of anhydride resulting from Example 7 (50 mg, 0.24 mmol) in anhydrous dichloromethane (0.5 ml) at room temperature were successively added pyridine (250 ml, 3.1 mmol), pyrrolidinopyridine (10 mg, 0.07 mmol) and cephalotaxine (76.4 mg, 0.24 mmol). After stirring at 18±5° C. for 48 hours, were successively added 1,3-dicyclohexylcarbodiimide (100 mg, 0.48 mmol), methanol (60 ml, 1.5 mmol), pyrrolidinopyridine (10 mg, 0.07 mmol) and toluene (1 ml). After stirring at 18±5° C. for 24 hours (with control of reaction in CCM), the reaction mixture was filtered and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (99: 1), silica (15-40 mm)) to provide expected product (12 mg, two diastereomers) contaminated with regioisomer* (two diastereomers) resulting from the opening of anhydride. The expected product thus obtained showed the following characteristics:

[0546]¹H NMR 400 MHz (CDCl₃)(d ppm, J Hz): 6.61 (1H, s, H-17*); 6.57 (1H, s, H-14*); 5.91 (J₃₋₄=9.8) and 5.84 (2H, 2d, H-3); 5.84 et 5.79 (2d, J_(AB)=1.4, OCH₂O); 5.84 and 5.82 (2d, J_(AB)=1.4, OCH₂O); 5.04 and 5.01 (1H, 2s, H-1); 3.79 and 3.78 (1H, 2d, J₄₋₃ =9.6, H-4); 3.70 and 3.65 (3H, 2s, OCH₃); 3.59 (3H, s, OCH₃); 3.15 (1H, m, H-11b); 3.09 (1H, m, H-8a); 2.94 (1H, m, H-10a); 2.58 (2H, m, H-8b+H-10b); 2.37 (1H, m, H-11a); 2.16 and 1.81 (2d, J_(AB)=14.4, CH₂CO₂); 2.13 and 1.66 (2d, J_(AB)=14.3, CH₂CO₂); 2.02 (1H, m, H-6_(A)); 1.88 (1H, m, H-6_(B)); 1.75 (2H, m, CH₂-7); 1.8-1.2 (6H, m, 3×CH₂); 1.11 and 1.02 (2s, 2×CH₃); 1.10 and 1.04 (2s, 2×CH₃).

[0547] *The regioisomer above-mentioned was also obtained from the following conditions:

[0548] To a stirred mixture of hemiester resulting from Example 6 method C (100 mg, 0.43 mmol) in anhydrous toluene (1 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (120 mg, 0.58 mmol). After stirring for 5 minutes, cephalotaxine (45 mg, 0.15 mmol) and pyrrolidinopyridine (21 mg, 0.14 mmol) were added. After stirring at 35° C. for 45 minutes, then at 8° C. for 15 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 mm) 4 g) to provide expected product (23 mg, 30%, two diastereomers). The product thus obtained showed the following characteristics:

[0549]¹H NMR 400 MHz (CDCl₃)(d ppm, J Hz): 6.61 and 6.58 (1H, 2s, H-17*); 6.57 and 6.53 (1H, 2s, H-14*); 5.89 and 5.86 (2d, J_(AB)=1.5, OCH₂O); 5.87 and 5.85 (2d, J_(AB)=1.5, OCH₂O); 5.76 (1H, d, J₃₋₄=9.4, H-3); 5.02 (1H, 2s, H-1); 3.73 and 3.72 (1H, 2d, J₄₋₃=9.4, H-4); 3.70 and 3.68 (3H, 2s, OCH₃); 3.69 and 3.65 (3H, 2s, OCH₃); 3.15 (1H, m, H-11a); 3.07 (1H, m, H-8a); 2.90 (1H, m, H-10a); 2.74 and 1.95 (2d, J_(AB)=15.3, CH₂CO₂); 2.56 (2H, m, H-8b+H-10b); 2.33 (1H, m, H-11a); 2.28 and 2.23 (2d, J_(AB)=15.4, CH₂CO₂); 2.16 (m, H-3′_(eq)); 1.97 (1H, m, H-6_(A)); 1.9-1.1 (5H, m, CH₂); 1.86 (1H, m, H-6_(B)); 1.73 (2H, m, CH₂-7); 1.14 (3H, s, CH₃); 1.03 (3H, s, CH₃).

EXAMPLE 10

[0550] Preparation of (−)-cephalotaxyl 2-methoxycarbonylmethyl-6,6-dimethyl-2 tetrahydropyrane carboxylate or anhydrohomoharringtonine, from tetrahydropyranecarboxylic acid resulting from Example 6:

[0551] 1°) Method via Mixed Anhydride

[0552] To a stirred mixture of hemiester resulting from Example 6 (50 mg, 0.22 mmol) in anhydrous toluene (1 ml) at room temperature was added triethylamine (dried over potassium hydroxide) (29.4 ml, 0.22 mmol) and 2,4,6-trichlorobenzoyl chloride (32.7 ml, 0.22 mmol). After stirring at 25° C. for 20 hours (with control of disappearing of starting acid in infra-red), 4-dimethylaminopyridine (29 mg, 0.24 mmol) was added, the reaction mixture was allowed to react for 5 minutes and cephalotaxine (16.5 mg, 0.05 mmol) was added. After stirring at 25° C. for 24 hours, the reaction mixture was filtered on paper and diluted with ether (5 ml). The resulting organic layer was successively washed with water (5 ml), with saturated sodium hydrogen carbonate solution (5 ml), with water again (5 ml), then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 mm)) to provide expected product (16 mg, 56%, two diastereomers). The product thus obtained showed identical characteristics to this obtained in Example 9.

[0553] 2°) Method Using DCC

[0554] To a stirred mixture of hemiester resulting from Example 6 (100 mg, 0.43 mmol) in anhydrous toluene (1 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (180 mg, 0.87 mmol). After stirring for 10 minutes, cephalotaxine (165 mg, 0.52 mmol) and pyrrolidinopyridine (77 mg, 0.52 mmol) were added. After stirring at 18±5° C. for 18 hours, was added ether, the reaction mixture was filtered on ground-glass filter, and the cake was washed with ether. The resulting organic layer was successively washed with 10% sodium hydrogen carbonate solution, with water, then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 mm) 9 g) to provide a solid (110 mg, 48%). The product thus obtained showed identical characteristics to this obtained in Example 9.

EXAMPLE 11

[0555] Preparation of (−)-cephalotaxyl (2′RS)-2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydropyrane carboxylate or anhydrohomoharringtonine, from ethylenic acid resulting from Example 6:

[0556] 1°) Method A: via Mixed Anhydride, Coupling with Cyclisation

[0557] To a stirred mixture of ethylenic ester resulting from Example 5 (50 mg, 0.22 mmol) in anhydrous toluene (1 ml) at room temperature was added triethylamine (dried over potassium hydroxide) (29 ml, 0.22 mmol) and 2,4,6-trichlorobenzoyl chloride (34 ml, 0.22 mmol). After stirring for 30 minutes (with control of disappearing of starting acid in infra-red), 4-dimethylaminopyridine (30 mg, 0.25 mmol) was added, the reaction mixture was allowed to react for 5 minutes and cephalotaxine (31 mg, 0.1 mmol) was added. After stirring at 18±5° C. for 65 hours, the reaction mixture was filtered on paper and diluted with ether (5 ml). The resulting organic layer was successively washed with water (5 ml), with saturated sodium hydrogen carbonate solution (5 ml), with water again (5 ml), then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 mm)) to provide expected product (46 mg, 96%, two diastereomers 40/60). The product thus obtained showed identical characteristics to this obtained in Example 9.

[0558] 2°) Method B: Using DCC, Coupling with Cyclisation

[0559] To a stirred mixture of ethylenic acid resulting from Example 5 (50 mg, 0.22 mmol) in anhydrous toluene (2 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (270 mg, 1.31 mmol). After stirring for 5 minutes, cephalotaxine (70 mg, 0.22 mmol) and pyrrolidinopyridine (32 mg, 0.22 mmol) were added. After stirring at 18±5° C. for 65 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 mm) 9 g) to provide a solid (40 mg, 35%). The product thus obtained showed identical characteristics to this obtained in Example 9.

EXAMPLE 12

[0560] Preparation of purified (−) cephalotaxine from total alkaloidic extract of Cephalotaxus sp

[0561] 1°) preparation of total alkaloids extract:

[0562] In a 30 liters-tank, crushed leaves (fresh or dry) of Cephalotaxus sp (10 kg) were mixed with methanol (201) and steed during 65 hours, then percolated (501). Solution was filtered and concentrated under vacuum to a volume of 51. Concentrated solution was acidified with a 6% aqueous solution of tartaric acid. Then hydro-alcoholic solution was washed by dichloromethane (5′ 51) for removing fatty materials and pigments. Aqueous solution was basified with aqueous ammonia (2.5%) until pH 9, then extracted with dichloromethane (5×5 l). After concentration under reduced pressure, crude alkaloids extract was recovered as a white crystalline solid (24.5 g). Cephalotaxine contain was 71% (HPLC).

[0563] 2°) Isolation and Chromatographic Purification of (−)-cephalotaxine from Crude Alkaloids Extract:

[0564] Above crude extract was dissolved in mobile phase (triethylamine (1.55/1000) in deionised water and orthophosphoric acid to adjust pH to 3. The solution was filtered then injected on a preparative high-performance liquid chromatograph equipped with axial compression and high pressure pump (stationary phase: n-octadecylsilane, 15 mm, porosity 100, 1 kg). Elution was performed at a flow rate of 0.2 l/min. Fractions contain was monitored by U.V. detector and TLC. Retained fraction were finally checked by HPLC then combined, alkalinised with 2.5% aqueous ammonia and extracted with dichloromethane (4×400 ml), After concentration under reduced pressure a resin was obtained which on trituration with methanol gave (−)-cephalotaxine (18 g) as a white crystalline solid (HPLC purity=99.8%). The product thus obtained showed the following characteristics

[0565] [α]_(D) ²⁰: −174,1 (c=0,20; CHCl₃)

[0566]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 6.68 (1H, s, H-17*); 6.65 (1H, s, H-14*); 5.91 and 5.90 (2H, 2d, J_(AB)=1.5, OCH₂O); 4.93 (1H, s, H-1); 4.77 (1H, dd, J₃₋₄=9.4, J_(3-OH)=3.4, H-3); 3.73 (3H, s, OCH₃); 3.68 (1H, d, J₄₋₃=9.4, H-4); 3.35 (1H, m, J_(AB)=14.3, J=12.2 and 7.9, H-11b); 3.08 (1H, m, J=9.1 and 4.9, H-8a); 2.92 (1H, td, J=11.6 and 7.1, H-10a); 2.59 (2H, m, H-8a +H-10a); 2.35 (1H, dd, J_(AB)=14.4, J=6.9, H-11a); 2.02 (1H, td, J_(AB)=12.1, J=9.7, H-6A); 1.87 (1H, m, J_(AB)=12.1, J=7.9 and 4.4, H-6_(B)); 1.74 (2H, m, CH₂-7); 1.62 (1H, d, J_(3-OH)=3.5, 3-OH).

EXAMPLE 13

[0567] Preparation of Lithium Alcoolate of (−) Cephalotaxine (Trapped like 3-O-acetyl Derivative)

[0568] 1°) Butyllithium Method

[0569] A commercial solution of butyllithium in hexane (0.44 ml, 1.6 M in hexane, 0.70 mmol) was added to a stirred mixture of (−)-cephalotaxine (200 mg, 0.63 mmol) in anhydrous tetrahydrofurane (6.8 ml). The reaction mixture was maintained at −60° C. for 20 minutes, then at −48° C. for 30 minutes, acetic anhydride (90 ml, 0.095 mmol) was added over a period of 8 minutes and the stirring was maintained at −48° C. for 20 minutes then at 0° C. for 1 hour. The mixture was quenched with saturated ammonium chloride solution (5 ml) then extracted with ethyl acetate (3′ 8 ml). The combined organic layers were washed with brine (15 ml) dried over magnesium sulfate and evaporated to dryness. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 mm) 6 g) to provide a white solid (60 mg, 26%). The product thus obtained showed the following characteristics:

[0570]¹H NMR 400 MHz (CDCl₃) (d ppm, J Hz): 6.60 (1H, s, H-17*); 6.57 (1H, s, H-14*); 5.89 and 5.86 (2H, 2d, J_(AB)=1.4, OCH₂O); 5.80 (1H, d, J₃₋₄=9.3, H-3); 5.05 (1H, s, H-1); 3.77 (1H, d, J₄₋₃=9.4, H-4); 3.72 (3H, s, OCH₃); 3.23 (1H, m, J_(AB)=14.3, J=12.3 and 7.9, H-11b); 3.08 (1H, m, H-8a); 2.92 (1H, td, J=11.5 and 7.1, H-10a); 2.57 (2H, m, H-8b+H-10b); 2.36 (1H, dd, J_(AB)=14.4, J=7.0, H-11a); 2.02 (1H, td, J_(AB)=12.1, J=9.7, H-6_(A)); 1.88 (1H, m, J_(AB)=12.1, J=8.0 and 4.0, H-6_(B)); 1.74 (2H, m, CH₂-7); 1.57 (3H, s, OAc).

[0571] 2°) Lithium bis-(trimethylsilyl)amide (LHDS) Method

[0572] A commercial solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane (0.95 ml, 0.95 mmol) was added to a stirred solution of (−)-cephalotaxine (200 mg, 0.63 mmol) in anhydrous tetrahydrofurane at −40° C. After stirring for 5 minutes, acetic anhydride (90 ml, 0.95 mmol) was added, and the reaction mixture was treated like method above-mentioned in 1°). The product thus obtained showed identical characteristics to this obtained above in butyllithium method.

[0573] 3°) Lithium Diisopropylamide (LDA) Method

[0574] A commercial solution of lithium diisopropylamide 2M in tetrahydrofurane (0.35 ml, 0.70 mmol) was added to a stirred solution of (−)-cephalotaxine (200 mg, 0.63 mmol) in anhydrous tetrahydrofurane (6.8 ml) at −60° C. over a period of 20 minutes After stirring at −60° C. for 20 minutes, then at −48° C. for 30 minutes, acetic anhydride (90 ml, 0.95 mmol) was added. The solution was stirred at −48° C. for 20 minutes, then at 0° C. for 1 hour and the reaction mixture was treated like method above-mentioned in 1°). The product thus obtained showed identical characteristics to this obtained above in butyllithium method.

[0575] 4°) Sodium Hydride Method

[0576] To a stirred mixture of sodium hydride (1.5 g) in freshly distilled dimethylformamide (3 ml) were added at −60° C. a solution of cephalotaxine (200 mg, 0.63 mmol) in dimethylformamide (3 ml) and acetic anhydride (90 ml, 0.95 mmol). After stirring at ambient temperature for 24 hours, the reaction mixture was treated at 0° C. with water (3 ml) and extracted with ether (3 5 ml). The combined organic layers were dried over magnesium sulfate and evaporated on vacuum. The product thus obtained showed identical characteristics to this obtained above in butyllithium method.

EXAMPLE 14

[0577] Preparation of (−)-cephalotaxyl 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydropyrane Carboxylate or Anhydrohomoharringtonine, via Lithium Alcoolate of Cephalotaxine: 

What is claimed is:
 1. A process for the preparation of sidechain-bearing cephalotaxane of the following formula and/or a salt thereof: Ω-CO—O—CTX where Ω (“omega”) is a representative radical of the chain terminal moiety and —CO— is the carbonyl of the ester group bonded to cephalotaxane; the Ω-CO— radical is corresponding: either to the following substituted heterocycloalkane formula:

where n ranges from 0 to 8; Z is oxygen or sulfur heteroatom, or NH; R⁵, R⁶ and R⁸ are independently hydrogen; hydrocarbon radical, saturated, unsaturated or aromatic, linear or branched and/or cyclic, optionally including heteroatom(s); R⁶ and R⁸ may be present in a cycle; oxygen ether bearing one of the former radicals; or to the following linear alkene formula:

where m ranges from 1 to 8; R⁵, R⁶ and R⁸ are as defined above; or to the following formula:

where n, R⁵, R⁶ and R⁸ are as defined above; Z and Q² are independently oxygen, NH or sulfur heteroatom; Q1 is carbon, silicium or phosphorus atom; R⁹ and R¹⁰ are independently hydrogen, alkoxy, hydrocarbon radical, optionally including heteroatom(s), saturated, unsaturated or aromatic, linear or branched and/or cyclic; R⁹ and/or R¹⁰ having the ability to be null or taken together to make an heteroatom and/or make a multiple bond with Q¹, R⁹ and R¹¹ having the ability to be null to make a multiple bond between the two atoms of carbon bearing them; and R¹¹ is hydrogen, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl or alkylcarbonyl; where —O—CTX is cephalotaxine moiety of the following formula or a salt thereof:

where p is equal to 1 or 2; the two types of radicals -Ω and —CTX above-mentioned being bonded with an ester bond —CO—O— the said process bringing together: either carboxylic acid with general formula Ω-CO—OH or a salt thereof; or an activated form of an acid with general formula Ω-CO—A or a salt thereof, with Ω-CO of the following formula:

where n, Z, R⁵, R⁶ and R⁸ are as defined above; where Ω-CO having the following formula:

m ranges from 1 to 8, Z, R⁵, R⁶ and R⁸ are as defined above; or Ω-CO having the following formula:

where n, Z, Q¹, Q², R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined above A represents: either cyclic anhydride of the following formula:

where n, R⁶ and R⁸ are as defined above; this reaction has been completed by methylation of the primary carboxyl thus formed with: either a hydroxyl group bearing cephalotaxane or a salt thereof of the formula H—O—CTX, where CTX are as defined above; or a metallic alcoxide of the formula M—O—CTX, where CTX are as defined above and M is a metal; or an activated form of its hydroxyl group of the formula Y—O—CTX, where —O—CTX is as defined above and Y is, either a leaving group to allow a negative charge on oxygen atom by cleavage between Y— and —O—CTX, or to allow a carbocation by cleavage between Y—O— and —CTX; with the possible presence of one or several reaction additives to form said sidechain-bearing cephalotaxane and/or a salt thereof.
 2. The process of claim 1, wherein Z is an oxygen atom and the cephalotaxane is a cephalotaxine of the following formula, or a salt thereof:

where R¹, R², R³ and R⁴ are independently hydrogen, hydroxyl group or alkoxide.
 3. The process of claim 2, wherein said cephalotaxane is cephalotaxine, or a salt thereof:

where R¹ is hydroxyl, R² is methoxyl, R³ and R⁴ are hydrogen.
 4. The process of claim 1 wherein R⁵ is hydrogen.
 5. The process of claim 1, wherein R⁵ is —CH₂—CO—O—Me.
 6. The process of claim 1, wherein n is 1 to 4, R⁶ and R ⁸are methyl.
 7. The process of claim 1, wherein n is 1 or 2, R⁶ is phenyl and R⁸ is hydrogen.
 8. The process of claim 1 wherein, R⁵ is —CH₂—CO—O—Me, said cephalotaxane is the former of claim 3, n is 0, Z is a nitrogen atom and R⁸ is hydrogen.
 9. The process of claim 1, wherein A is Ω-CO—O—radical.
 10. The process of claim 1, wherein A is halide.
 11. The process of claim 1, wherein A is a radical of compound Ω-CO—A having the ability to generate cleavage of the bond between carbonyl group and substituent A of Ω-CO—A to provide Ω-CO⁺ and A⁻.
 12. The process of claim 1, wherein A is a radical selected from substituents: méthoxyformyloxy of formula MeOCOO—, trifluoroacétyloxy of formula CF₃COO—, alkylsulfonoxy of formula RSO₃—, phosphoxy of formula (RO)₂PO—, halophosphoxy of formula ROP(Cl)O—, trialkylsilyloxy of formula R₃ SiO—, diméthyl-formamidinium chloride of formula:

or acyloxy-pyridinium bromide of the formula:

formulas wherein R is alkyl.
 13. The process of claim 1, wherein A is a 2,4,6-trichlorobenzoyloxy radical.
 14. The process of claim 13, wherein the reagent of formula Ω-CO—A when A is 2,4,6-trichlorobenzoyloxy radical is obtained by contacting an acid Ω-CO—OH, as defined according to claim 1, with 2,4,6-trichlorobenzoyl chloride in presence of one or more O-acylation additives.
 15. The process of claim 1, wherein radical A corresponds to the following formula:


16. The process of claim 15, wherein the reagent of formula Ω-CO—A is obtained by contacting an acid Ω-CO—OH, as defined according to claim 1, with carbonyl-diimidazole in presence of a strong base.
 17. The process of claim 16, wherein the strong base is an alkoxide.
 18. The process of claim 1, wherein a coupling additive is present, comprising a substituted carbodiimide and/or a basic additive such as tertiary amine.
 19. The process of claim 18, wherein the substituted carbodiimide is selected from cyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC) and chlorhydrate of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.
 20. The process of claim 1, wherein the cephalotaxine alcoxide corresponds to the following formula: M—O—CTX, is obtained by contacting a cephalotaxine of the following formula: H—O—CTX with metal, an amidure, a metallic hydride or an alkyl-metal.
 21. The process of claim 1, wherein M is an alkaline metal.
 22. A sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

where n ranges from 0 to 8; Z is oxygen, NH or sulfur heteroatom; R⁵, R⁶ and R⁸ are independently hydrogen; hydrocarbon radical, saturated, unsaturated or aromatic, linear or branched and/or cyclic, optionally including heteroatom(s); oxygen ether bearing one of the former radicals; —O—CTX is cephalotaxine moiety of the following formula or a salt thereof:

where p is equal to 1 or 2; the two types of radicals -Ω and —CTX above-mentioned being bonded with an ester bond —CO—O— except for compounds where Z is oxygen atom and, 1°)n is 2 or 3, and simultaneously R⁶=R⁸=methyl and R⁵ is —CH₂COOMe, 2° n is 2 and simultaneously R⁶=R⁸=methyl and R⁵ is OMe or hydroxyl; 3° n is 3 and simultaneously R⁶ is hydroxyl, when R⁸ is methyl and R⁵ is a —CH₂CO₂CH₃ radical.
 23. A sidechain-bearing cephalotaxane corresponding to the following formula:


24. The process of claim 1, wherein when the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, of the following formula:

where n, R⁵, R⁶, R⁸, CTX and Z are as defined according to claim 1; the said chain is open with an agent and/or a protonic electrophilic radical E in aqueous or not aqueous medium, to provide an intermediate compound of the following formula:

where n, CTX, R⁵, R⁶ and R⁸are as defined above, E is either hydrogen or the provisionally or definitively fixed eletrophilic radical; the aforementioned intermediate compound may be attacked with an agent or a nucleophilic radical Z′, deliberately added or possibly present in the medium, and when the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, of the following formula:

where n, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined according to claim 1, and Z′ is an heteroatom; the said chain is open by hydrolysis or carefully solvolysis with possibly presence of activation and/or opening additive; to provide an open sidechain-bearing cephalotaxane of the following formula:

where n, CTX, R⁵, R⁶ and R⁸ are as defined according to claim 1; Z′ is: either an halogen or an heteroatom bearing a hydrogen or a radical R¹¹ such as defined accordint to claim 1; or an hydrogen, hydrocarbon radical, the said radical bearing or not heteroatom(s), saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, or heterocycloalkyl.
 25. The process of claim 24, wherein the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, of the following formula:

where n, R⁸, R⁶, R⁵, CTX and Z are defined according to the claim 1, the said chain is open by treatment with a solution of hydrobromic acid in acetic acid, in an halogenated solvent, followed by in situ hydrolysis to provide, without isolation of the intermediate, a sidechain-bearing cephalotaxane of the following formula:


26. The process according to claim 1, wherein said acids correspond to the following formula: Ω-CO—OH the said formula equivalent to racemic mixture containing compounds of the formulas (+)-Ω-CO—OH and (−)-Ω-CO—OH wherein (+)-Ω-CO—OH represents its dextrogyre enantiomer and (−)-Ω-CO—OH represent its levogyre enantiomer. were obtained a) by contacting said racemic mixture or one of the activated forms of the formula Ω-CO—A which is as defined according to claim 1; the said racemic mixture or said activated form generating respectively: either an anion corresponding to the formula (Ω-CO—O)⁻; or a cation corresponding to the formula (Ω-CO)⁺; with a pure enantiomeric form of chiral entity, said “resolution agent” symbolized by Δ* (delta stella), having the ability to form: either a stable combination, by covalent bonding; or an easily reversible labil combination, by hydrogen bonding or by hydrophobic interaction; or intermediate lability bonding by electrostatic interaction; to provide a diastereomeric mixture of Ω-CO—O-Δ* and de Ω-CO-Δ*; b) then by physical separation of the mixture of two diastereomers or two complex compounds or more generally of two new entities physically and/or chemically different then obtained; c) then by regeneration and finally separation of each one of enantiomers of the generic formula Ω*-CO—OH, where Ω* (<<oméga stella>>) represents the generic symbol of the same chiral radical in the either one or the other pure enantiomeric forms corresponding to the following formulas (+)-Ω-CO—OH and (−)-Ω-CO—OH which are as defined above.
 27. The process of claim 26, wherein Ω-CO— is a radical corresponding to the following formula:


28. The process of claim 26, wherein the stable combination is represented by an ester of the following formula Ω-CO—O-Δ*, said stable combination obtained by contacting acid with a chiral alcohol corresponding to the formula HO-Δ*.
 29. The process of claim 26, wherein the stable combination is represented by an amide corresponding to the either one or the other formulas Ω-CO—NH-Δ* or Ω-CO—N-Δ*, said stable combination obtained by contacting acid with primary or secondary chiral amine corresponding to formulas H₂N-Δ* or NNisΔ*.
 30. The process of claim 26, wherein the stable combination is represented by an thioester of the following formula Ω-CO—S-Δ*, said stable combination obtained by contacting acid with a chiral thiol corresponding to the formula HS-Δ*.
 31. The process of claim 26, wherein the ionic combination is represented by a salt prepared by contacting an acid with a chiral amine corresponding to one of the following formulas: Ω-CO—O⁻ [NH-Δ*]⁺Ω-CO—O⁻[NH₂-Δ*]⁺Ω-CO—O⁻ [NH₃-Δ*]⁺
 32. The process of claim 26, wherein the bringing into play of a labile bonding based combination is achieved in the form of chromatography with the help of a chiral stationary phase.
 33. The process of claim 26, wherein the bringing into play of an interatomic or intermolecular labile bonding based combination, within crystalline latice, is achieved in the form of fractionated crystallization initiated by a chiral precursor.
 34. The process of claim 28, wherein chiral alcohol HO-D* is (−)-quinine corresponding to the following formula:


35. The process of claim 28, wherein the chiral alcohol HO-A* is (−)- or (+)-methyl mandelate corresponding to the following formulas:


36. The process of claim 28, wherein the chiral alcohol HO-D* is (−)- or (+)-menthol corresponding to the following formulas:


37. The process of claim 31, wherein the chiral amine H₂N-A* is (−)- or (+)-ephedrine corresponding to the following formulas:


38. The process of claim 1, wherein the carboxylic acid is: either the tertiary heterocycloalcane carboxylic acid corresponding to the following formula:

where n, Z, R⁵, R⁶ and R⁸ are as defined according to claim 1; the said acid is obtained by treatment in aprotic or protic solvant, eventually in the presence of cyclization additive and/or dehydrating agent, the said treatment eventually supported with physical carrying of the water formed. or open tertiary ethylenic acid corresponding to the following formula:

where m, Z, R⁵, R⁶and R⁸ are as defined according to claim 1; or open tertiary ethylenic acid corresponding to the following formula:

where m ranges from t to 8, Z, R⁵, R⁶ and R⁸ are as defined according to claim 1, R¹², is not a CTX radical according to claim 1, represents R⁵ and/or a protective group of acids and/or a chiral group; then R¹² is removed later, either just by saponification, or by hydrogenolysis, or by a method to remove protective groups of acids.
 39. The process of claim 38, wherein in the absence of a cyclization additive, the reaction of cyclization is conducted by heating.
 40. The process of claim 38, wherein the cyclization additive is a protic acid or an aprotic acid, included in immobilized form.
 41. The process of claim 38, wherein the acid is sulfonic acid or formic acid.
 42. The process of claim 38, wherein Z is an oxygen atom.
 43. The process of claim 1, wherein said cephalotaxane H—O—CTX is a cephalotaxine of the following formula, or a salt thereof, or a diastereo isomer thereof:

where R¹³ and R¹⁴ are independently hydrogen, hydroxyl or alkoxide and R¹⁵ to R¹⁸ and R²⁰ to R²⁶ are independently hydrogen, hydroxyl, thiol, halogen, alcoxy, alkylthio group, or alkoxide, or R¹⁵, R¹⁷ and R¹⁸ can further independently represent OR¹⁹, R¹⁹ being hydrogen or an hydrocarbon radical.
 44. The process of claim 43, wherein R²⁰ to R²⁶ are hydrogen, R¹³ and R¹⁴ are independently hydroxyl or hydrogen, R¹⁵ to R¹⁸ are defined as in claim
 43. 45. The process of claim 44, wherein R¹⁶ and R²⁰ to R²⁶ are hydrogen, R¹³ is hydroxyl, R¹⁴ is hydrogen, R¹⁵, R¹⁷ and R¹⁸ are independently OR¹⁹, R¹⁹ being alkyl, alkenyl, alkynyl, cycloalkyl, cycloaklenyl, aryl or heteroalkyl, said radicals including or not heteroatom(s).
 46. The process of claim 45, wherein H—O—CTX is drupacine of formula:

or a salt thereof.
 47. The process of claim 2, where R¹ and R⁴ are hydroxyl, R² is hydrogen and R³ is methoxyl, wherein said cephalotaxane H—O—CTX is 11-hydroxy-cephalotaxine, or a salt thereof, of formula:


48. A sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

where n is included between 0 and 8; Z is oxygen, NH or sulfur heteroatom; R⁵, R⁶ and R⁸ are independently hydrogen, hydrocarbon radical, saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl, of said radical including or not heteroatom(s), oxygen ether bearing one of the former radicals; CTX is as defined according to claim
 43. 49. The sidechain-bearing cephalotaxane of claim 48, where R⁵ is CH₂CO₂Me and R⁶=R⁸=methyl, corresponding to the following formula and/or a salt thereof:


50. The sidechain-bearing cephalotaxane of claim 49, where CTX is a drupacine radical as defined according to claim
 46. 51. The sidechain-bearing cephalotaxane of claim 49, where CTX is a 11-hydroxy-caphalotaxine radical as defined according to claim
 47. 52. A sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

where n, CTX, R⁵, R⁶ and R⁸ are as defined according to claim 48 and Z′ is defined according to claim 24; except for compounds where Z′ is hydrogen and simultaneously R⁶=R⁸=methyl and R⁵ is CH₂CO₂Me and, 1°) n is 2 and CTX is a drupacine radical as defined according to claim 46; 2°) n is 2 or 3, and CTX is a 11-hydroxy-cephalotaxine radical as defined according to claim
 47. 53. The sidechain-bearing cephalotaxane of claim 52, where Z′ is hydroxyl.
 54. The sidechain-bearing cephalotaxane according to claim 53, where R⁵ is CH₂CO₂Me and R⁶=R⁸=methyl and CTX is a drupacine radical as defined according to claim 46, corresponding to the following formula and/or a salt thereof: 